Antimicrobial Electrodeposited Silver-Containing Calcium Phosphate Coatings.

Biocompatible antimicrobial coatings may enhance the function of many orthopedic implants by combating infection. Hydroxyapatite is a choice mineral for such a coating as it is native to bone and silver would be a possible antimicrobial agent as it is also commonly used in biomedical applications. The aim of the research is to develop a silver-containing calcium phosphate (Ag/Ca-P) coating via electrochemical deposition on titanium substrates as this allows for controlled coating buildup on complex shapes and porous surfaces. Two different deposition approaches are explored: one-step Ag/Ca-P(1) deposition coatings, containing silver ions as microsized silver phosphate particles embedded in the Ca-P matrix; and via a two-step method (Ag/Ca-P(2)) where silver is deposited as metallic silver nanoparticle on the Ca-P coating. The Ag/Ca-P(1) coating displays a bacterial reduction of 76.1 ± 8.3% via Ag-ion leaching. The Ag/Ca-P(2) coating displays a bacterial reduction of 83.7 ± 4.5% via contact killing. Interestingly, by preincubation in phosphate-buffered saline solution, bacterial reduction improves to 97.6 ± 2.7 and 99.7 ± 0.4% for Ag/Ca-P(1) and Ag/Ca-P(2) coatings, respectively, due to leaching of formed AgClx(x-1)- species. The biocompatibility evaluation indicates that the Ag/Ca-P(1) coating is cytotoxic towards osteoblasts while the Ag/Ca-P(2) coating shows excellent compatibility. The electrochemical deposition of highly bactericidal coatings with excellent biocompatibility will enable us to coat future bone implants even with complex or porous structures.

Mechanical and biological properties of electrodeposited calcium phosphate coatings.

Calcium phosphate (CaP) coatings were electrochemically deposited on titanium substrates. By increasing the electrodeposition time (from 1 to 30 min), the coating thickness increases but also the surface morphology of the CaP coatings is greatly affected going from smooth to plate-like, featuring elongated plates, ribbon-like and finally sharp needle structures. Micro-stretch tests reveal that, regardless of the coating morphology and thickness, the electrodeposited CaP coatings have strong adhesion with the titanium substrates and their failure mode is cohesive failure. The effects of different morphologies on cellular behavior such as adhesion, viability, proliferation, and osteogenic gene expression were studied. The surface morphology of CaP coatings has a remarkable effect on cell attachment, proliferation, and viability. A smooth surface results in better adhesion of the cells, whereas the presence of sharp needles and ribbons on rough surfaces restricts cell adhesion and consequently cell proliferation and viability. The improved cell adhesion and viability on the smoother surface can be attributed to the higher contact area between the cell and the coating, while the needle-like morphology inflicts damage to the cells by physically disrupting the cell wall. There is no significant difference in the level of osteoblast gene expression when osteosarcoma cells are cultured on coatings with different morphologies. Our study provides crucial insights into the optimum electrodeposition procedures for CaP coating formation leading to both good cell-material interaction and sufficient mechanical properties. This can be achieved with relatively thin coatings produced by short electrodeposition times.

3D impedimetric sensors as a tool for monitoring bacterial response to antibiotics.

The presence of antimicrobial contaminants like antibiotics in the environment is a major concern because they promote the emergence and the spread of multidrug resistant bacteria. Since the conventional systems for the determination of bacterial susceptibility to antibiotics rely on culturing methods that require long processing times, the implementation of novel strategies is highly required for fast and point-of-care applications. Here the development and characterization of a novel label-free biosensing platform based on a microbial biosensor approach to perform antibiotic detection bioassays in diluted solution is presented. The microbial biosensor is based on a three-dimensional interdigitated electrode array (3D-IDEA) impedimetric transducer with immobilized E. coli bacteria. In 3D-IDEA to increase the sensitivity to superficial impedance changes the electrode digits are separated by insulating barriers. A novel strategy is employed to selectively immobilize bacteria in the spaces over the electrode digits between the barriers, referred to here as trenches, in order to concentrate bacteria, improve the reproducibility of the E. coli immobilization and increase the sensitivity for monitoring bacterial response. For effective attachment of bacteria within the trenches an initial anchoring layer of a highly charged polycation, polyethyleneimine (PEI), was used. To facilitate immobilization of bacteria within the trenches and prevent their deposition on top of the barriers an important novelty is the use of poly(N-isopropylmethacrylamide) p(NIPMAM) microgels working as antifouling agents, deposited on top of the barriers by microcontact printing. The reported microbial biosensor approach allows the bacterial response to ampicillin, a bacteriolytic antibiotic, to be registered by means of impedance variations in a rapid and label-free operation that enables new possibilities in bioassays for toxicity testing.

Recombinant Newcastle disease viruses with targets for PCR diagnostics for rinderpest and peste des petits ruminants.

Since February 1st 2011, rinderpest (RP) has been officially declared eradicated worldwide. National authorities have been requested to destroy all their RP related materials. Nonetheless, their national reference laboratories performing real time reverse transcription polymerase chain reaction assays (PCR diagnostics) need RP positive control samples, since some countries still prefer to maintain diagnostic capability for RP for several reasons. In the future, a similar situation will arise for peste des petits ruminants (PPR) as the ambition has been expressed to eradicate PPR. Anticipating on this, we intended to perform qualified PCR diagnostics without use of infectious RPV or PPRV. Therefore, Newcastle disease virus (NDV) with small RNA inserts based on RPV or PPRV sequences were generated and used as positive control material. Recombinant NDVs (recNDVs) were differentially detected by previously established PCR diagnostics for RPV or PPRV. Both recNDVs contain a second PCR target showing that additional targets in NDV are feasible and would increase the diagnostic sensitivity by use of two PCR assays. RecNDV with small PCR targets is not classified as RPV or PPRV containing material, and can be used to mimic RPV or PPRV. Using these recNDVs as virus positive material contributes to the ambition of worldwide eradication, while qualified PCR diagnostics for these OIE-listed diseases remains operational.

Effect of Vagus Nerve Integrity on Short and Long-Term Efficacy of Antireflux Surgery.

OBJECTIVES: Vagus nerve injury is a feared complication of antireflux surgery (ARS) that may negatively affect reflux control. The aim of the present prospective study was to evaluate short-term and long-term impact of vagus nerve injury, evaluated by pancreatic polypeptide response to insulin-induced hypoglycemia (PP-IH), on the outcome of ARS. METHODS: In the period from 1990 until 2000, 125 patients with gastroesophageal reflux disease (GERD) underwent ARS at a single center. Before and 6 months after surgery, vagus nerve integrity testing (PP-IH), 24-h pH-monitoring, gastric emptying, and reflux-associated symptoms were evaluated. In 2014, 14-25 years after surgery, 110 patients were contacted again for evaluation of long-term symptomatic outcome using two validated questionnaires (Gastrointestinal Symptom Rating Scale (GSRS) and GERD-Health Related Quality of Life (HRQL)). RESULTS: Short-term follow-up: vagus nerve injury (PP peak ≤47 pmol/l) was observed in 23 patients (18%) 6 months after fundoplication. In both groups, a comparable decrease in reflux parameters and symptoms was observed at 6-month follow-up. Postoperative gastric emptying was significantly delayed in the vagus nerve injury group compared with the vagus nerve intact group. Long-term follow-up: patients with vagus nerve injury showed significantly less effective reflux control and a higher re-operation rate. CONCLUSIONS: Vagus nerve injury occurs in up to 20% of patients after ARS. Reflux control 6 months after surgery was not affected by vagus nerve injury. However, long-term follow-up showed a negative effect on reflux symptom control and re-operation rate in patients with vagus nerve injury.

Bluetongue virus with mutated genome segment 10 to differentiate infected from vaccinated animals: a genetic DIVA approach.

Bluetongue virus (BTV) includes 24 serotypes and recently even more serotypes are proposed. Mass vaccination campaigns highlight the need for differential diagnostics in vaccinated populations. Bluetongue disease is routinely diagnosed by serological and virological tests by which differentiation infected from vaccinated animals (DIVA principle) is not possible. Real time PCR tests preferably detect all BTV serotypes (panBTV PCR tests). These PCR tests operate as frontline test to detect new BTV incursions. However, highly sensitive panBTV PCR tests can also detect currently applied inactivated and modified-live vaccines. Here, BTV with eight silent mutations in segment 10 (Seg-10) was generated by reverse genetics. This BTV mutant is not detected by a Seg-10 panBTV PCR test (genetic DIVA). Thus, inactivated BT vaccine with this mutated Seg-10 will avoid false positive PCR results post vaccination, whereas BTV infected animals can be positively diagnosed with the accompanying Seg-10 panBTV PCR test (DIVA-test) far beyond the infectious period.

Preliminary mapping of non-conserved epitopes on envelope glycoprotein E2 of Bovine viral diarrhea virus type 1 and 2.

Bovine viral diarrhea virus (BVDV) belongs together with Classical swine fever virus (CSFV) and Border disease virus (BDV) to the genus Pestivirus in the Flaviviridae family. BVDV has been subdivided into two different species, BVDV1 and BVDV2 based on phylogenetic analysis. Subsequent characterization of both strains revealed major antigenic differences. Because the envelope glycoprotein E2 is the most immunodominant protein for all pestiviruses, the present study focused on epitope mapping by constructing chimeric BVDV type 1 and 2 E2 genes in expression plasmids. These plasmids with chimeric E2-genes were transfected in SK6 cells and transient expression was studied by immunostaining with a panel of MAbs specific for E2 of BVDV1 or BVDV2, resulting in the localization of type-specific antigenic domains at similar regions. These results indicate that E2 glycoproteins of both BVDV types exhibit a comparable antigenic structure, but with type specific epitopes. In addition, the antigenic resemblance with envelope glycoprotein E2 of Classical swine fever virus is discussed.

Bluetongue virus serotype 8 (BTV-8) infection reduces fertility of Dutch dairy cattle and is vertically transmitted to offspring.

In 2007, BTV-8 re-emerged for the second year in the Netherlands and caused morbidity and increased mortality in cattle herds. In addition, cattle farmers reported reduced fertility in their cows. For this study, fifteen herds that were not vaccinated were selected. These were matched to 10 vaccinated herds by geographic region. At the start of the study, in July 2008, all cattle in the non-vaccinated herds >1 year old were sampled. All seronegative cows entered the study program and blood samples from these cows were tested for antibodies against BTV-8 in an ELISA. Cows were sampled at intervals of three weeks and sampling was stopped once a cow tested seropositive. Sampling ceased in all remaining cows in December 2008. Newborn calves originating from infected dams or from vaccinated dams were tested by PCR for BTV-8. Fertility data were obtained from the Royal Dutch Cattle Syndicate (CRV). Multi-level generalized latent and linear models were used for analyses. In 2008, 185 (17.2%) out of 1,074 initially seronegative non-vaccinated cattle seroconverted and were assumed to be infected with BTV-8. Infected cows were 5 (95% CI: 1.9-14.3) times more likely to return for insemination within 56 days after first insemination. In addition, these cows needed 1.7 (95% CI: 1.4-2.0) times more inseminations for an assumed pregnancy, and needed 2.5 (95% CI: 2.4-2.6) times more days between first and last insemination compared to the period prior to seroconversion and compared to cows not infected by BTV-8 in 2008. No association between BTV-8 infection and the chance to abort between 100 and 260 days after last insemination was found. In total, 48 calves originating from infected cows were tested by PCR for the presence of BTV-8. Ten (20.8%) out of these 48 calves were born PCR-positive. None of 256 calves from vaccinated dams tested PCR-positive. Further, cows infected during the second half of gestation had a 15.5 times (95% CI: 1.3-190.4) higher chance of a PCR-positive newborn calf compared to cows infected in the first half of gestation. This study showed that BTV-8 has a negative effect on fertility of dairy cattle.

Laparoscopic Nissen fundoplication after failed EsophyX fundoplication.

BACKGROUND: Reflux control may be ineffective in a substantial number of patients after endoluminal EsophyX fundoplication for gastro-oesophageal reflux disease. Subsequent laparoscopic Nissen fundoplication (LNF) might be required to relieve symptoms. The aim of this study was to evaluate the outcome of LNF after previous EsophyX fundoplication. METHODS: EsophyX failure was defined as recurrence or persistence of typical symptoms, with or without anatomical failure of the wrap or persisting pathological oesophageal acid exposure. Consecutive patients who underwent LNF after failed EsophyX fundoplication were identified. Symptomatic outcome was obtained by standardized questionnaire, and objective outcome by endoscopy, oesophageal manometry and pH monitoring. RESULTS: Eleven patients were included. During LNF, intraoperative gastric perforation occurred in two patients and one developed a subphrenic abscess after operation. Daily heartburn was present in one patient after LNF and three had troublesome daily dysphagia. General quality of life after LNF was not significantly better than that before EsophyX fundoplication. Oesophageal acid exposure was normalized in all patients after surgery. Oesophagitis was absent after LNF in all except one patient who had persisting grade A oesophagitis. CONCLUSION: Symptomatic and objective reflux control are satisfactory after LNF for a failed EsophyX procedure. Previous EsophyX fundoplication, however, is associated with a risk of gastric injury during LNF and a relatively high rate of postfundoplication dysphagia.

Evaluation of an indirect ELISA for detection of antibodies in bulk milk against bluetongue virus infections in the Netherlands.

After the introduction of bluetongue virus serotype 8 (BTV-8) in western Europe in 2006, an indirect ELISA for detection of serogroup-specific antibodies against BTV in serum samples was validated for individual milk samples by the Central Veterinary Institute and the Animal Health Service in the Netherlands (Kramps et al., 2008). In order to develop a cost-effective monitoring tool, we now have evaluated this ELISA also for use in bulk milk. Therefore, bulk milk samples and individual milk samples were collected from 92 herds in the affected southern region in the Netherlands in 2007, before the start of the vaccination campaign. In addition, bulk milk samples collected from 88 herds before the bluetongue introduction in 2006 ("historically negative" samples) have been tested. With these results ROC analyses were performed and herd specificity and herd sensitivity of the bulk milk ELISA were estimated. All "historically negative" bulk milk samples were negative in the ELISA, with a mean S/P ratio of 10 ± 0.8%. The herd sensitivity and herd specificity of the ELISA in bulk milk samples depend on the cut-off that is chosen. In order to detect a within-herd-prevalence of 1%, the optimal cut-off S/P ratio 13% was found. A few herds with one or two milk-positive animals would then be missed. The specificity will be 100%. A within-herd-prevalence of 10% can be detected with 100% sensitivity at a cut-off S/P ratio of 96%. In conclusion, the indirect ELISA in bulk milk samples is a very specific and sensitive test which can be implemented in monitoring and surveillance systems in unvaccinated populations.

Questionnaire survey about the motives of commercial livestock farmers and hobby holders to vaccinate their animals against Bluetongue virus serotype 8 in 2008-2009 in the Netherlands.

After a massive epidemic of Bluetongue virus serotype 8 (BTV-8) among ruminants in 2006-2007 in the European Union (EU), the Netherlands started a voluntary emergency vaccination campaign in May 2008, subsidized by the EU. At the start of a new campaign in 2009, without subsidized vaccination, we investigated by mail survey the motives of farmers and hobby holders to vaccinate against BTV-8 in 2008 and 2009. Mean vaccine uptake in 2008 was: 73% in sheep, 71% in cattle, 43% in goat farms and 67% in hobby holdings. Top-5 motives pro-vaccination were: prevention of production loss; subsidized vaccination; recommendation by practitioner; welfare reasons; contribution to the eradication campaign. Top-5 motives against vaccination were: vaccination costs; absence of clinical BT-problems; presumed low infection risk; balance between vaccination costs and loss without vaccination; bad experience with earlier vaccination campaigns. Willingness to vaccinate was significantly lower in 2009: 42% in sheep, 58% in cattle, 19% in goat farms and 49% in hobby holdings. Measures to stimulate vaccination among those that did not want to vaccinate in 2009 were: subsidized vaccination; possibility to vaccinate their own animals; more information on efficacy/safety of vaccine and why animals had to be vaccinated again; availability of a BT vaccine combined with vaccine(s) against other diseases.

A decade of washing out common bile duct stones with papillary balloon dilatation as a one-stage procedure during laparoscopic cholecystectomy.

BACKGROUND: Laparoscopic cholecystectomy is the treatment of choice for the management of cholecystolithiasis. For the management of choledocholithiasis, a number of options exist. The effectiveness of washing out common bile duct stones with laparoscopic transcystic papillary balloon dilatation (LTPBD) in patients undergoing laparoscopic cholecystectomy (LC) as a one-stage procedure was evaluated. METHODS: Retrospectively, the files of 63 patients treated with LTPBD in a one-stage procedure undergoing laparoscopic cholecystectomy between December 1996 and December 2006 were studied. RESULTS: Fifty-three patients were treated successfully in a one-stage procedure, seven patients were treated in two steps with an endoscopic retrograde cholangiopancreatography (ERCP) postoperatively, and in three cases a conversion to open surgery was required. The median operation time was 128 min, and the median hospital stay was 4 days. No patients developed postoperative pancreatitis. In one case contrast leakage from the common bile duct was detected. It was the only complication directly related to the LTPBD. There were no postoperative deaths. CONCLUSIONS: We consider the wash out of common bile duct stones after LTPBD in a one-stage procedure to be an easy to do and safe operation with great results. Cooperation with an intervention radiologist and application of an angioplastic dilatation dotter balloon catheter are the keys to success in this procedure. In our hospital, it is the treatment of choice for choledocholithiasis associated with cholelithiasis.

Vertical transmission of bluetongue virus serotype 8 virus in Dutch dairy herds in 2007.

In February 2008, evidence was found for transplacental infection of bluetongue virus serotype 8 (BTV-8) in PCR negative, seropositive heifers in Northern Ireland originating from the Netherlands. The relevance of this route of transmission was studied in Dutch cow-calf combinations in the Netherlands of which the calves were born in the same time period of the year as the calves from the exported heifers, the first quarter of 2008. Blood samples were tested from 385 cows and their calves, housed in 43 dairy farms that became naturally infected with BTV-8 for the first time in 2007. All calves were at least 10 days old at the moment of first testing. In total 229 cows tested seropositive for BTV-8. Eight of these cows were still PCR positive. Out of the 229 seropositive cows, 37 calves (16.2%; 95% CI: 11.4-21.0) were tested PCR positive in the first sample taken in April 2008. In the first week of June, 34 out of the 37 PCR positive calves were still available for resampling. Three calves were still PCR positive; one was 5 months old, the other two were 3 months old. One month later, in the first week of July, all initially PCR positive calves, including the three still tested positive 1 month earlier, were PCR negative. We showed that BTV-8 can be vertically transmitted from cow to calf and can result in healthy looking viraemic calves remaining PCR positive for up to 5 months. These PCR positive calves could play a role in the epidemiology, and in particular in overwintering of BT. However, further investigations are needed to evaluate the importance of this route of transmission.

Performance of clinical signs to detect bluetongue virus serotype 8 outbreaks in cattle and sheep during the 2006-epidemic in The Netherlands.

The performance of clinical signs as a diagnostic test for the detection of BTV-8 outbreaks during the 2006-epidemic in The Netherlands was evaluated by constructing and analysing receiver operating characteristic (ROC) curves. The area under the ROC curve of the BT-associated clinical signs in cattle was 0.77. An optimal efficient test (maximising both sensitivity and specificity) in cattle herds combined a sensitivity (Se) of 67% with a specificity (Sp) of 72%, comprising the following clinical signs: ulcerations and/or erosions of oral mucosa or erosions of lips/crusts in or around nostrils or oedema of the nose or hyperaemic/purple coloration of tongue, tongue protrusion or coronitis or apathy/tiredness or muscle necrosis, stiffness of limbs or loathing or refusal to move, prostration or torticollis or anoestrus. The area under the ROC curve of the BT-associated clinical signs in sheep was 0.81. The optimal efficient test in sheep flocks combined a Se of 76% with a Sp of 72%, comprising the following clinical signs: ulcerations of oral mucosa or serous nasal discharge or erosions/ulceration of tongue mucosa or hypersensitivity of the skin or muscle necrosis, stiffness of limbs or coronitis or grinding of teeth or salivation or weakness/paresis.

[First outbreak of bluetongue in goats in The Netherlands]. / Eerste bluetongue-uitbraak bij geiten in Nederland: beschrijving van de klinische verschijnselen en differentieeldiagnose.

For the first time, bluetongue has been diagnosed in goats in the Netherlands and in Northwest-Europe. On the 17th of August 2006, bluetongue was for the first time diagnosed in sheep and a little later in cattle in The Netherlands. The clinical symptoms, diagnostics and differential diagnosis of bluetongue (BT) in goats in the Netherlands are described. The most obvious clinical signs were an acute drop in milk production and high fever (up to 42 degrees C). Clinical signs were less obvious than usually seen for clinically diseased sheep and cattle. A few goats showed oedema of the lips and the head, some nasal discharge and scabs on the nose and lips. Further erythema of the skin of the udder and small subcutaneous hemorrhages were seen. Just like one year ago, for the very first suspicion of bluetongue in Northwest-Europe, a good collaboration between practitioners, specialists of the Animal Health Service (GD Deventer), the Specialist Team of the Food and Consumer Product Safety Authority (VWA), and the Central Institute for animal Disease Control (CIDC-Lelystad) in The Netherlands, led to the first and rapid notification and confirmation of the suspicion of bluetongue.