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

Dutch national mastitis survey. The effect of herd and animal factors on somatic cell count

The purpose of the national mastitis survey was to collect information on the prevalence and causes of bovine subclinical mastitis in the Netherlands. Milk samples were collected once from 10 336 cows in a stratified random selection of herds (n = 227) in the Netherlands during the years 1985-1986. Results showed that 84.2 % of the cows were free from mastitis. Ten per cent of all udder quarters were infected, and 3.7 % of these were infected with Staphy(ococcus aureus, the main udder pathogen. Statistical analysis based on a 'split-plot' model was used to analyse the effect of herd factors and animal factors on somatic cell counts (SCC). Several factors significant­ly influenced SCC: breed, season, geographical region, type of housing, and the use of teat disin­fection. The effect of herd and animal factors on SCC of milk samples of individual cows was calculated as deviation from the geometric mean cell count of the standard cow (222 000/ml) and presented as the excpected SCC per cow. The interaction of parity x stage of lactation x infec­tion status also significantly influenced SCC. On the basis of expected SCC of uninfected cows correction factors were calculated for individual cows with various parities and at various stages of lactation. We conclude that the use of these correction factors can improve the analysis of SCC in the diagnosis of mastitis. <br/

Knoflook remt App

De Animal Sciences Group van Wageningen UR heeft in opdracht van biologische varkenshouders onderzocht of het mogelijk is om in plaats van antibiotica, knoflook te gebruiken voor de bestrijding van longontsteking door de bacterie Actinobacillus pleuropneumoniae (App). Uit de resultaten blijkt dat Allyl Methyl Sulfi de (AMS), een omzettingsproduct van knoflook, de groei van App remt

An experimental <i>Toxoplasma gondii </i>dose response challenge model to study therapeutic or vaccine efficacy in cats

High numbers of Toxoplasma gondii oocysts in the environment are a risk factor to humans. The environmental contamination might be reduced by vaccinating the definitive host, cats. An experimental challenge model is necessary to quantitatively assess the efficacy of a vaccine or drug treatment. Previous studies have indicated that bradyzoites are highly infectious for cats. To infect cats, tissue cysts were isolated from the brains of mice infected with oocysts of T. gondii M4 strain, and bradyzoites were released by pepsin digestion. Free bradyzoites were counted and graded doses (1000, 100, 50, 10), and 250 intact tissue cysts were inoculated orally into three cats each. Oocysts shed by these five groups of cats were collected from faeces by flotation techniques, counted microscopically and estimated by real time PCR. Additionally, the number of T. gondii in heart, tongue and brains were estimated, and serology for anti T. gondii antibodies was performed. A Beta-Poisson dose-response model was used to estimate the infectivity of single bradyzoites and linear regression was used to determine the relation between inoculated dose and numbers of oocyst shed. We found that real time PCR was more sensitive than microscopic detection of oocysts, and oocysts were detected by PCR in faeces of cats fed 10 bradyzoites but by microscopic examination. Real time PCR may only detect fragments of T. gondii DNA without the presence of oocysts in low doses. Prevalence of tissue cysts of T. gondii in tongue, heart and brains, and anti T. gondii antibody concentrations were all found to depend on the inoculated bradyzoite dose. The combination of the experimental challenge model and the dose response analysis provides a suitable reference for quantifying the potential reduction in human health risk due to a treatment of domestic cats by vaccination or by therapeutic drug application

Prevalence of <i>Mycobacterium avium</i> in Slaughter Pigs Based on Serological Monitoring Results and Bacteriological Validation

Mycobacterium avium (MA) is a potential food safety hazard in pigs. Blood samples of slaughtered pigs in the Netherlands and Germany were tested for the presence of MA antibodies to estimate the serological prevalence in the tested population. In the Dutch and German population 1.0% and 1.7% samples were positive, and 0.5% and 17.4% of the herds were at risk for having a MA infection respectively. The validity of the applied MA-ELISA was evaluated under field conditions. The specificity of the MA-ELISA was high (>98.4%). The average herd sensitivity was 18%. In the affected herds on average 50% of the animals were tested bacteriological positive for MA. It can be concluded that serological screening for the presence of MA antibodies is capable of identifying pig populations that are at risk for a MA infection

Type 2 Endoleak With or Without Intervention and Survival After Endovascular Aneurysm Repair

Objective: The aims of the present study were to examine the impact of type 2 endoleaks (T2EL) on overall survival and to determine the need for secondary intervention after endovascular aneurysm repair (EVAR). Methods: A multicentre retrospective cohort study in the Netherlands was conducted among patients with an infrarenal abdominal aortic aneurysm (AAA) who underwent EVAR between 2007 and 2012. The primary endpoint was overall survival for patients with (T2EL+) or without (T2EL-) a T2EL. Secondary endpoints were sac growth, AAA rupture, and secondary intervention. Kaplan–Meier survival and multivariable Cox regression analysis were used. Results: A total of 2 018 patients were included. The median follow up was 62.1 (range 0.1 – 146.2) months. No difference in overall survival was found between T2EL+ (n = 388) and T2EL- patients (n = 1630) (p =.54). The overall survival estimates at five and 10 years were 73.3%/69.4% and 45.9%/44.1% for T2EL+/T2EL- patients, respectively. Eighty-five of 388 (21.9%) T2EL+ patients underwent a secondary intervention. There was no difference in overall survival between T2EL+ patients who underwent a secondary intervention and those who were treated conservatively (p =.081). Sac growth was observed in 89 T2EL+ patients and 44/89 patients (49.4%) underwent a secondary intervention. In 41/44 cases (93.1%), sac growth was still observed after the intervention, but was left untreated. Aneurysm rupture occurred in 4/388 T2EL patients. In Cox regression analysis, higher age, ASA classification, and maximum iliac diameter were significantly associated with worse overall survival. Conclusion: No difference in overall survival was found between T2EL+ and T2EL- patients. Also, patients who underwent a secondary intervention did not have better survival compared with those who did not undergo a secondary intervention. This study reinforces the need for conservative treatment of an isolated T2EL and the importance of a prospective study to determine possible advantages of the intervention

<i>Streptococcus suis</i> infections in pigs: use of virulence-associated markers in diagnostics and vaccines = <i>Streptococcus suis</i> infecties bij varkens: het gebruik van virulentiekenmerken in diagnostiek en vaccins

Streptococcus suis is an important pig pathogen which is mainly associated with meningitis, arthritis and septicaemia. Control of the disease is hampered by the lack of effective vaccines and the lack of reliable diagnostic tests with high specificity and sensitivity. The development of these tools is complicated by the number of existing serotypes, by the fact that we still lack knowledge of the factors responsible for virulence and protection, and by the fact that strains may vary in virulence. Therefore, research focused on the identification of virulence-associated markers that discriminate between virulent and less virulent or avirulent isolates, has gained considerable interest in recent years. The aim of the investigations described in this thesis, was to test whether these virulence-associated markers could be used in diagnostic assays for the detection of S. suis infections and/or for use in vaccines to protect against the disease. In previous work, muramidase-released protein (MRP) and extracellular-factor protein EF were identified as markers of virulence in serotype 1 and 2 strains. In other serotypes the production of MRP and EF, and their potential importance for bacterial virulence has not been investigated. Therefore, we determined the serotypes as well as MRP and EF phenotypes for a collection of S. suis strains isolated from diseased pigs in seven European countries. Overall, S. suis serotype 2 appeared to be most prevalent (32Œ followed by serotype 9 (20€and serotype 1 (12Ž EF-positive strains, were found in serotype 1 (66Œ 2 (71€and 14 (85€strains. Variants of MRP (MRP* or MRPs) were found in nearly all serotypes. A high percentage (81€of the serotype 9 strains belonged to the MRP*EF- phenotype. For the detection of pigs carrying virulent serotype 2 strains, serotype 1, 1/2, 7, 9 and 14 strains, Multiplex PCR tests have been developed. In Multiplex PCR 1, three DNA targets, based on the S. suis serotype 1 (and 14), 7 and 9 specific capsular polysaccharide (cps) genes, were amplified. In Multiplex PCR II, two other targets, based on the serotype 2 (and 1/2) specific cps gene and the epf gene encoding the EF-protein, were amplified. The evaluation of these PCRs for use on tonsillar specimens of diseased pigs demonstrated that the assays were highly specific and sensitive. For the development of protective vaccines, the efficacy of a MRP and EF vaccine applied in pigs was tested. Pigs were vaccinated twice and challenged intravenously with virulent S. suis serotype 2 strains. At challenge, pigs vaccinated with MRP and EF were protected against infection and disease. Pigs vaccinated with either MRP or EF were less well protected. Apparently the combination of both proteins is necessary to obtain full protection. The protective efficacy of an avirulent, non-encapsulated isogenic mutant of S. suis serotype 2 was determined in pigs, and compared with the efficacy of the capsulated wild-type strain. Vaccinations were with formalin-killed cells of the wild-type (WT-BAC), formalin-killed cells of the non-encapsulated mutant (CM-BAC) or with the live non-encapsulated mutant (CM-LIVE) strain. Pigs were challenged intravenously with the homologous, wild-type S. suis serotype 2 strain. The results demonstrated that, as expected, the formalin-killed cells of WT-BAC induced complete protection in pigs against mortality and morbidity after challenge. The formalin-killed cells of CM-BAC induced complete protection against mortality, but only partial protection against morbidity. The CM-LIVE vaccine induced only partial protection, both against mortality and morbidity. These findings indicate that CPS and other bacterial components of WT-BAC are probably essential for full protection against homologous challenge

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