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Young piglets are very susceptible to infections and therefore vaccination early after birth can help to decrease the disease incidence on swine farms. However, vaccination during the neonatal period is a challenge, because vaccination of newborn pigs is in general less successful compared to adult pigs. The innate and adaptive immune system are still developing during early-life and this results in qualitative and quantitative differences between the immune responses of newborn or neonatal pigs (< 1-week-old) and adult or immunocompetent (> 8-week-old) pigs after vaccination. Research has indicated that effective immuno-stimulators (adjuvants) in adult vaccines may not fulfil the requirements to stimulate the neonatal immune system effectively. Therefore, age-group-adapted vaccine formulations may be necessary to enhance the success of vaccination at an early age. The aim of this thesis was to investigate immune responses in neonatal and adult pigs after Toll-like receptor (TLR) stimulation to increase our understanding of the porcine (neonatal) immune system and the immune responses after vaccination in pigs of different age-groups. In addition to conventional intramuscular (i.m.) vaccination, skin vaccination was used as a delivery route, because skin has the potential to increase vaccine efficacy due to its abundance of antigen presenting cells (APCs). Porcine reproductive and respiratory syndrome virus (PRRSV) was selected as vaccine-antigen, because this is an endemic and important disease in the swine farming. In addition, to date no effective inactivated PRRSV-vaccine has been developed yet.In chapter 2, we studied dendritic cells (DCs) and showed that neonatal porcine DCs derived from blood were composed of the same DC subsets and DC proportions as their adult counterparts, namely: plasmacytoid DCs (pDC), and two types of conventional DCs (cDC1 and cDC2). After TLR1/2 or TLR9 stimulation peripheral blood mononuclear cells (PBMCs) and DCs were activated in b

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Contains fulltext : 171208.pdf (publisher's version ) (Open Access), BACKGROUND: Streptococcus pneumoniae, a Gram-positive bacterium carried in the human nasopharynx, is an important human pathogen causing mild diseases such as otitis media and sinusitis as well as severe diseases including pneumonia, meningitis and sepsis. There is a strong resemblance between the anatomy, immunology and physiology of the pig and human species. Furthermore, there are striking similarities between S. suis pathogenesis in piglets and S. pneumoniae pathogenesis in humans. Therefore, we investigated the use of piglets as a model for pneumococcal colonization and invasive disease. RESULTS: Intravenous inoculation of piglets with an invasive pneumococcal isolate led to bacteraemia during 5 days, showing clear bacterial replication in the first two days. Bacteraemia was frequently associated with fever and septic arthritis. Moreover, intranasal inoculation of piglets with a nasopharyngeal isolate led to colonization for at least six consecutive days. CONCLUSIONS: This demonstrates that central aspects of human pneumococcal infections can be modelled in piglets enabling the use of this model for studies on colonization and transmission but also on development of vaccines and host-directed therapies. Moreover this is the first example of an animal model inducing high levels of pneumococcal septic arthritis.

To study Schmallenberg virus (SBV) excretion in bovine semen after experimental infection, two bulls were inoculated subcutaneously with a SBV isolate (1 ml Vero cell culture 106 TCID50). After inoculation (at day 0), semen was collected daily from both animals for 21 days and samples were tested for SBV by qRT–PCR assay. At 24 days post-inoculation both animals were subjected to necropsy and the genital organs and lymph nodes draining these organs were also tested for SBV RNA (qRT–PCR). After SBV infection both animals in the study showed viraemia (qRT–PCR) with fever and diarrhoea. SBV RNA could be detected in semen from both animals. The highest SBV RNA concentrations in semen were found in the first week (days 4–7 post-inoculation) but concentrations were relatively low (Ct values 30–39). Viable SBV was only isolated from blood samples and not from semen or genital tissues.

Background - Maternal antibodies can interfere with foot-and-mouth disease vaccination. In this study we determined whether intratypic heterologous vaccination could help to improve herd immunity. Results - In unvaccinated calves, a half-life of maternal antibodies of 21 days was determined. At two weeks of age, calves without maternal antibodies showed a good antibody response against both vaccines used in the trial, while in calves with maternal antibodies no antibody response to homologous vaccination (A Turkey 14/98) but a limited antibody response to intratypic heterologous vaccination (A22 Iraq) was observed. Conclusion - Two weeks old calves without maternal antibodies respond well to vaccination, but when emergency vaccination is carried out in a region that uses prophylactic vaccination, using an intratypic heterologous vaccine strain may improve the immunity in calves with maternal antibodies

Background: Schmallenberg virus (SBV) has swept through the major part of Europe in the period 2011–2013. A vaccine against SBV has been developed and may be a possible preventive instrument against infection. Presently, there is no data available to refute the assumption that natural SBV infection results in long-term immunity. In that respect, it is of interest to know how long (protecting) virus-neutralizing antibodies are present in naturally infected animals. New-born calves acquire passive immunity from their dams by ingestion and absorption of antibodies present in colostrum, which can block the production of serum antibodies when vaccine is administered to calves with maternally derived antibodies. In that respect, it is useful to know how long it takes for maternal antibodies against SBV to disappear in young animals born from infected dams. Results: Longitudinal whole-herd serological monitoring using virus neutralization test (VNT) indicated that 80% of adult dairy cows still had measurable antibodies against SBV at least 24 months after the estimated introduction of the virus into the herd. Median 2Log VNT titer of the adult dairy cows (=1 year) dropped from 8.6 to 5.6 in a period of 17 months. Median 2Log VNT maternal antibodies titers of calves sampled within 30 days after birth was 8. Calves lost their maternally-derived antibodies after 5–6 months. There was a definite positive relationship between the VNT titer of the dam and the VNT titer of the corresponding calf (age = 30 days) of dam-calf combinations sampled on the same day: the higher the VNT titer of the dam, the higher the VNT titer (maternal antibodies) of the calf. Conclusions: Our field data support the assumption that natural SBV infection in adult cows results in persistence of specific antibodies for at least two years. Based on the observed decay of maternally-derived antibodies in calves, it is presumed safe to vaccinate calves against SBV at an age of approximately 6 months

Background Highly pathogenic avian influenza (HPAI) viruses pose a potential human health threat as they can be transmitted directly from infected poultry to humans. During a large outbreak of HPAI H7N7 virus among poultry in The Netherlands in 2003, bird to human transmission was confirmed in 89 cases, of which one had a fatal outcome. Methods To identify genetic determinants of virulence in a mammalian host, we passaged an avian H7N7/03 outbreak isolate in mouse lungs and evaluated the phenotype of the mouse-adapted variant in animal models and in vitro. Results Three passages in mouse lungs were sufficient to select a variant that was highly virulent in mice. The virus had a MLD50 that was >4.3 logs lower than that of its non-lethal parental virus. Sequence analysis revealed a single mutation at position 627 in PB2, where the glutamic acid was changed to a lysine (E627K). The mouse-adapted virus has this mutation in common with the fatal human case isolate. The virus remained highly pathogenic for chickens after its passage in mice. In ferrets, the mouse-adapted virus induced more severe disease, replicated to higher titers in the lower respiratory tract and spread more efficiently to systemic organs compared with the parental virus. In vitro, the PB2 E627K mutation had a promoting effect on virus propagation in mammalian, but not in avian cells. Conclusions Our results show that the E627K mutation in PB2 alone can be sufficient to convert an HPAI H7N7 virus of low virulence to a variant causing severe disease in mice and ferrets. The rapid emergence of the PB2 E627K mutant during mouse adaptation and its pathogenicity in ferrets emphasize the potential risk of HPAI H7N7 viruses for human health.

The European Union 1997 Directive, stipulating that veal calves should be fed a minimum of 50 to 250 g of fibrous feed from 8 to 20 wk of age, is vague. A fibrous feed ration maximum of 250 g has been implicated in welfare issues, namely the occurrence of abnormal oral behaviors and poor gastrointestinal health. Past research suggests that this amount is insufficient to prevent the development of abnormal oral behaviors and enabling good rumen development. Different sources and particle sizes of roughage could lead to very different welfare outcomes. In a 3 × 2 × 2 factorial design, 240 group-housed calves (10 ± 1 d; 46.1 ± 0.1 kg) were fed different roughage sources (straw, maize silage, or maize cob silage; the latter 2 were dried and provided no extra moisture compared with straw) in 2 amounts (250 or 500 g of dry matter per day), and 2 particle sizes (chopped or ground). Roughage was supplemented to milk replacer (MR) from 2 wk after arrival. In addition, 60 calves were fed 1 of 3 additional control treatments: MR only (n = 20), MR plus an iron supplement (n = 20), or MR plus ad libitum hay (n = 20). Oral behaviors were recorded using instantaneous scan sampling at 2-min intervals for 2 h in 3 periods per day, at 12 and 22 wk of age. Calves were slaughtered at 24 wk of age and rumen and abomasal health parameters were recorded. Limited provision of straw resulted in behavior comparable with that from unlimited provision of hay, with reduced tongue playing and oral manipulation of the environment, as well as increased chewing compared with diets with no roughage supplement. Straw prevented ruminal hairballs, but impaired rumen development and increased abomasal damage. A higher ration of roughage increased chewing (12 wk), decreased oral manipulation of the trough (12 and 22 wk) and the pen (22 wk), and increased rumen weight. However, more roughage led to increased abomasal damage for certain parameters. Longer feed particles had no obvious benefits for behavior

Streptococcus uberis is a highly prevalent causative agent of bovine mastitis, which leads to large economic losses in the dairy industry. The aim of this study was to examine the host response during acute inflammation after experimental challenge with capsulated Strep. uberis. Gene expression in response to Strep. uberis was compared between infected and control quarters in 3 animals. All quarters (n=16) were sampled at 16 different locations. Microarray data showed that 239 genes were differentially expressed between infected and control quarters. No differences in gene expression were observed between the different locations. Microarray data were confirmed for several genes using quantitative PCR analysis. Genes differentially expressed due to early Strep. uberis mastitis represented several stages of the process of infection: (1) pathogen recognition; (2) chemoattraction of neutrophils; (3) tissue repair mechanisms; and (4) bactericidal activity. Three different pathogen recognition genes were induced: ficolins, lipopolysaccharide binding protein, and toll-like receptor 2. Calgranulins were found to be the most strongly upregulated genes during early inflammation. By histology and immunohistochemistry, we demonstrated that changes in gene expression in response to Strep. uberis were induced both in infiltrating somatic milk cells and in mammary epithelial cells, demonstrating that the latter cell type plays a role in milk production as well as immune responsiveness. Given the rapid development of inflammation or mastitis after infection, early diagnosis of (Strep. uberis) mastitis is required for prevention of disease and spread of the pathogen. Insight into host responses could help to design immunomodulatory therapies to dampen inflammation after (early) diagnosis of Strep. uberis mastitis. Future research should focus on development of these early diagnostics and immunomodulatory components for mastitis treatment.

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Background Streptococcus suis is a zoonotic pathogen that causes infections in young piglets. S. suis is a heterogeneous species. Thirty-three different capsular serotypes have been described, that differ in virulence between as well as within serotypes.Results In this study, the correlation between gene content, serotype, phenotype and virulence among 55 S. suis strains was studied using Comparative Genome Hybridization (CGH). Clustering of CGH data divided S. suis isolates into two clusters, A and B. Cluster A isolates could be discriminated from cluster B isolates based on the protein expression of extracellular factor (EF). Cluster A contained serotype 1 and 2 isolates that were correlated with virulence. Cluster B mainly contained serotype 7 and 9 isolates. Genetic similarity was observed between serotype 7 and serotype 2 isolates that do not express muramidase released protein (MRP) and EF (MRP-EF-), suggesting these isolates originated from a common founder. Profiles of 25 putative virulence-associated genes of S. suis were determined among the 55 isolates. Presence of all 25 genes was shown for cluster A isolates, whereas cluster B isolates lacked one or more putative virulence genes. Divergence of S. suis isolates was further studied based on the presence of 39 regions of difference. Conservation of genes was evaluated by the definition of a core genome that contained 78% of all ORFs in P1/7.Conclusions In conclusion, we show that CGH is a valuable method to study distribution of genes or gene clusters among isolates in detail, yielding information on genetic similarity, and virulence traits of S. suis isolates.

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.

Background Streptococcus suis is a zoonotic pathogen that causes infections in young piglets. S. suis is a heterogeneous species. Thirty-three different capsular serotypes have been described, that differ in virulence between as well as within serotypes.Results In this study, the correlation between gene content, serotype, phenotype and virulence among 55 S. suis strains was studied using Comparative Genome Hybridization (CGH). Clustering of CGH data divided S. suis isolates into two clusters, A and B. Cluster A isolates could be discriminated from cluster B isolates based on the protein expression of extracellular factor (EF). Cluster A contained serotype 1 and 2 isolates that were correlated with virulence. Cluster B mainly contained serotype 7 and 9 isolates. Genetic similarity was observed between serotype 7 and serotype 2 isolates that do not express muramidase released protein (MRP) and EF (MRP-EF-), suggesting these isolates originated from a common founder. Profiles of 25 putative virulence-associated genes of S. suis were determined among the 55 isolates. Presence of all 25 genes was shown for cluster A isolates, whereas cluster B isolates lacked one or more putative virulence genes. Divergence of S. suis isolates was further studied based on the presence of 39 regions of difference. Conservation of genes was evaluated by the definition of a core genome that contained 78% of all ORFs in P1/7.Conclusions In conclusion, we show that CGH is a valuable method to study distribution of genes or gene clusters among isolates in detail, yielding information on genetic similarity, and virulence traits of S. suis isolates.

Item does not contain fulltext, The aim of this study is the development and evaluation of a serodiagnostic assay for Mycobacterium avium (MA). After screening MA lipid fractions in an ELISA format, a polar lipid fraction was selected as antigen because of its superior recognition by serum antibodies in experimentally infected pigs. The resulting MA-ELISA was evaluated as an alternative for detection of MA infection by traditional pathological examination of pig lymph nodes for granulomatous lesions by meat inspectors. By comparing with bacteriological examination, the MA-ELISA showed significantly better sensitivity (69%) as compared to pathological examination (31%) in experimentally infected pigs. The MA-ELISA also appeared significantly more specific in a set of serum samples from MA negative pigs: only 1 out of these 153 serum samples reacted positive, whereas 99 (65%) of these had displayed false positive results by detection of lymph nodes lesions that appeared not to be associated with MA (Komijn et al., 2007). The MA-ELISA was subsequently evaluated using serum samples from two farms with pigs known to be infected with MA. Bacteriological examination of the sub-maxillary and mesenteric lymph nodes showed that 56% (103/184) and 35% (41/117) of the pigs, respectively were positive for MA in these farms. In the first farm, 16% (29/184) of the pigs tested positive in MA-ELISA and 31% (57/184) by pathological examination. On the contrary, in the second farm, more pigs tested positive 17% (15/117) in MA-ELISA with 8% (9/117) positivity by pathological examination. Taking the results on both farms together, the sensitivity of the MA-ELISA was 14% and the specificity 83%, whereas the sensitivity of the pathological examination was 31% and the specificity 86%. For practical reasons use of a serological test as the MA-ELISA may be preferred over pathological or bacteriological examinations. Our studies in experimentally infected and negative "field" sera indicate that the MA-ELISA is significantly