Protection of pigs against challenge with virulent <i>Streptococcus suis</i> serotype 2 strains by a muramidase-released protein and extracellular factor vaccine

The efficacy of a muramidase-released protein (MRP) and extracellular factor (EF) vaccine in preventing infection and disease in pigs challenged either with a homologous or a heterologous Streptococcus suis serotype 2 strain (MRP EF ) was compared with the efficacy of a vaccine containing formalin-killed bacterin of S suis serotype 2 (MRP EF ). The enhancement of the immune response by different adjuvants (a water-in-oil emulsion [wo] and an aluminium hydroxide-based adjuvant [AH]) and their side effects were also studied. The MRP and EF were purified by affinity chromatography. Pigs were vaccinated twice at three weeks and six weeks of age and challenged intravenously with virulent S suis serotype 2 strains (MRP EF ) at eight weeks of age. At challenge, the pigs vaccinated with MRP EF/WO had high anti-MRP and anti-EF titres and were protected as effectively as pigs vaccinated with wo-formulated vaccines with bacterin. Eight of the nine pigs survived the challenge and almost no clinical signs of disease were observed. The titres obtained with the MRP EF/AH vaccine were low and only two of the five pigs were protected. Pigs vaccinated with either MRP or EF were less well protected; three of the four pigs died after challenge but the clinical signs of disease were significantly less severe than those observed in the placebo-vaccinated pigs. The protective capacity of the bacterin/AH vaccine was very low, and the mortality among these pigs was as high as in the placebo-vaccinated pigs (80 per cent). Postmortem histological examination revealed meningitis, polyserositis and arthritis in the clinically affected pigs. The results demonstrate that a subunit vaccine containing both MRP and EF, formulated with the wo adjuvant, protected pigs against challenge with virulent S suis type 2 strains

Dutch National Mastitis Survey. The value of bulk milk cellcounts in diagnosing bovine mastitis

Bulk milk samples of 227 at random selected herds and quarter milk samples of 10336 cows ofthese herds were collected once. Bulk milk somatic cell counts (BMSCCs), which are estimatedevery four weeks, being part of the Dutch Milk Quality Control Programme, were used to calculatethree derivative cell counts: the C3 count, the Cl3 count and the Ci8 count. The correlationof these counts with the prevalence of mastitis in herds was determined. Correlation coefficientsvaried from 0.42 to 0.62. BMSCC and the C3 were neither highly sensitive (maximum sensitivity 75%) nor predictive (maximum predictive value 70 %) in identifying herds with a high prevalenceof mastitis. At the cell count threshold C = 500, 55-76 % herds with a high prevalence ofsubclinical mastitis were not detected by the C3. Thirty-six percent of the herds with a C3 exceedingthe threshold of C = 500 and 42 % of the herds with a C3 exceeding the threshold of c =400 could not be classified as mastitis problem herds. Both BMSCC and C3 had higher sensitivitybut lower specificity and predictive value at a threshold of C = 400 than at the threshold of c= 500. BMSCC and the C3 could reasonably specifically (84-99 %) diagnose herds with a lowprevalence of mastitis. Because of their low sensitivity and predictive value it would not be justifiedto assume a high prevalence of subclinical mastitis on farms based on BMSCC and the C3,without further evidence. Direct bulk milk cell counts and their derivatives appeared unsuitablein determining the prevalence of mastitis in individual herds

756-1 Non Surgical Septum Reduction: A New Treatment for Hypertrophic Obstructive Cardiomyopathy (HOCM)

In patients with HOCM and marked intraventricular gradients resistant to conventional drug treatment with beta blockers and/or Verapamil surgical resection of the muscular septal bulge has been advocated. We have investigated a new catheter treatment in 5 patients with HOCM and significant LV ouflow tract gradients. All patients were in class 3 NYHA with angina and shortness of breath. Intraventricular gradients were measured with transeptally introduced Brockenbrough catheters in the LV inflow tract and arterial catheters in the aortic root. All patients were studied at rest, during the Valsalva manoeuvre, after nitrates and after Isoproterenol infusion. The measurements were repeated during balloon occlusion of the first major septal branch of the left anterior descending coronary artery. — In all patients the resting intraventricular gradient was reduced to less than 20mm/Hg and provocative testing (nitrates and post extrasystolic potentation) failed to create typical increments. The longest inflation time was 30 minutes. Three patients had Verapamil 0.5mg injected through the angioplasty balloon which resulted in a longer lasting gradient/reduction after deflation of the balloon.–After informed consent, 2 patient had 3–5ml of desiccated alcohol infused through the inflated balloon catheter in order to devitalise the offending myocardium. This resulted in a CK elevation up to 2,500 units and permanent abolition of the intraventricular gradient accompanied by marked clinical improvement.From these preliminary observations we conclude that non surgical septum ablation maybe a promising new technique for the treatment of HOCM. Further studies are warranted

Pre-Absorbed Immunoproteomics: A Novel Method for the Detection of Streptococcus suis Surface Proteins

Streptococcus suis serotype 2 (SS2) is a zoonotic pathogen that can cause infections in pigs and humans. Bacterial surface proteins are often investigated as potential vaccine candidates and biomarkers of virulence. In this study, a novel method for identifying bacterial surface proteins is presented, which combines immunoproteomic and immunoserologic techniques. Critical to the success of this new method is an improved procedure for generating two-dimensional electrophoresis gel profiles of S. suis proteins. The S. suis surface proteins identified in this study include muramidase-released protein precursor (MRP) and an ABC transporter protein, while MRP is thought to be one of the main virulence factors in SS2 located on the bacterial surface. Herein, we demonstrate that the ABC transporter protein can bind to HEp-2 cells, which strongly suggests that this protein is located on the bacterial cell surface and may be involved in pathogenesis. An immunofluorescence assay confirmed that the ABC transporter is localized to the bacterial outer surface. This new method may prove to be a useful tool for identifying surface proteins, and aid in the development of new vaccine subunits and disease diagnostics

Differences in the Population Structure of Invasive Streptococcus suis Strains Isolated from Pigs and from Humans in the Netherlands

Streptococcus suis serotype 2 is the main cause of zoonotic S. suis infection despite the fact that other serotypes are frequently isolated from diseased pigs. Studies comparing concurrent invasive human and pig isolates from a single geographical location are lacking. We compared the population structures of invasive S. suis strains isolated between 1986 and 2008 from human patients (N = 24) and from pigs with invasive disease (N = 124) in the Netherlands by serotyping and multi locus sequence typing (MLST). Fifty-six percent of pig isolates were of serotype 9 belonging to 15 clonal complexes (CCs) or singleton sequence types (ST). In contrast, all human isolates were of serotype 2 and belonged to two non-overlapping clonal complexes CC1 (58%) and CC20 (42%). The proportion of serotype 2 isolates among S. suis strains isolated from humans was significantly higher than among strains isolated from pigs (24/24 vs. 29/124; P<0.0001). This difference remained significant when only strains within CC1 and CC20 were considered (24/24 vs. 27/37,P = 0.004). The Simpson diversity index of the S. suis population isolated from humans (0.598) was smaller than of the population isolated from pigs (0.765, P = 0.05) indicating that the S. suis population isolated from infected pigs was more diverse than the S. suis population isolated from human patients. S. suis serotype 2 strains of CC20 were all negative in a PCR for detection of genes encoding extracellular protein factor (EF) variants. These data indicate that the polysaccharide capsule is an important correlate of human S. suis infection, irrespective of the ST and EF encoding gene type of S. suis strains

Lysozyme Resistance in Streptococcus suis Is Highly Variable and Multifactorial

Background: Streptococcus suis is an important infectious agent for pigs and occasionally for humans. The host innate immune system plays a key role in preventing and eliminating S. suis infections. One important constituent of the innate immune system is the protein lysozyme, which is present in a variety of body fluids and immune cells. Lysozyme acts as a peptidoglycan degrading enzyme causing bacterial lysis. Several pathogens have developed mechanisms to evade lysozyme-mediated killing. In the present study we compared the lysozyme sensitivity of various S. suis isolates and investigated the molecular basis of lysozyme resistance for this pathogen. Results: The lysozyme minimal inhibitory concentrations of a wide panel of S. suis isolates varied between 0.3 to 10 mg/ml. By inactivating the oatA gene in a serotype 2 and a serotype 9 strain, we showed that OatA-mediated peptidoglycan modification partly contributes to lysozyme resistance. Furthermore, inactivation of the murMN operon provided evidence that additional peptidoglycan crosslinking is not involved in lysozyme resistance in S. suis. Besides a targeted approach, we also used an unbiased approach for identifying factors involved in lysozyme resistance. Based on whole genome comparisons of a lysozyme sensitive strain and selected lysozyme resistant derivatives, we detected several single nucleotide polymorphisms (SNPs) that were correlated with the lysozyme resistance trait. Two SNPs caused defects in protein expression of an autolysin and a capsule sugar transferase. Analysis of specific isogenic mutants, confirmed th