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The presence and severity of lung lesions recorded post-mortem is commonly used as an indicator to assess the prevalence of respiratory problems in batches of bovines. In the context of a welfare monitoring based on on-farm measures, the recording of clinical signs on calves at the farm would be more convenient than the recording of lung lesions at slaughter. The aim of the present study was to investigate the relationship between clinical respiratory signs at farm and post-mortem analyses of lung lesions observed at slaughter in veal calves. If clinical signs were a good predictor of lung lesions it could be possible to integrate only those measures in a welfare monitoring system. One-hundred-and-seventy-four batches of calves were observed 3 times: at 3 and 13 weeks after arrival of the calves at the unit and at 2 weeks before slaughter. For each batch a maximum of 300 calves was observed and the proportions of calves showing abnormal breathing, nasal discharge and coughing were recorded. Post-mortem inspection was carried out on a sample of lungs belonging to calves from the observed batches. Each examined lung was classified according to a 4-point scale for pneumonia from healthy lung (score 0) to severe lesions (score 3). The clinical signs recorded infra vitam were significantly correlated with moderate and severe lung lesions for observations at 13 weeks and 2 weeks before slaughter and the level of the correlation was highly variable (r(sp) from 0.16 to 0.40). Receiver operating characteristic (ROC) curves were created and the area under the curves showed that batches with a high proportion of lungs with moderate or severe lesions could not be accurately detected by the three clinical signs of respiratory disorders. These results suggest that both clinical signs and post-mortem inspection of lung lesions must be included in a welfare monitoring schemes for veal calves.

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.

Lung diseases in pigs are among the most important health problem in pig husbandry, and generally treated with antimicrobials. To reduce the amount of antimicrobials used in pigs, we tested the preventive effects of freeze dried garlic added to feed on an infection with Actinobacilluspleuropneumoniae (APP). Thirty male pigs of about seven weeks of age were aerosol challenged with APP serotype 2. Fifteen pigs received 5% (w/w) garlic added to their diet (Garlic group), from two days prior to infection until four days after infection. The others received the a standard diet (Control group). There was no difference in the number of pigs with symptoms of lung diseases. 48 hours after infection, the number of white blood cells had significantly increased in the Control group but not in the Garlic group. In the Control group a positive correlation (p

Due to possible presence and spread of contagious animal viruses via natural sausage casings the international trade in these food products is subject to veterinary and public health requirements. In order to manage these restrictions we determined the effect of casing preservation on four highly contagious viruses for livestock: foot-and-mouth-disease virus (FMDV), classical swine fever virus (CSFV), swine vesicular disease virus (SVDV) and African swine fever virus (ASFV). We used an in vitro 3D collagen matrix model in which cells, infected with the four different viruses were embedded in a bovine collagen type I gel matrix and treated with either saturated salt (NaCl) or phosphate supplemented saturated salt at four different temperatures (4, 12, 20 and 25 °C) during a period of 30 days. The results showed that all viruses were faster inactivated at higher temperatures, but that stability of the various viruses at 4 °C differed. Inactivation of FMDV in the 3D collagen matrix model showed a clear temperature and treatment effect on the reduction of FMDV titres. At 4 and 12 °C phosphate supplemented salt showed a very strong FMDV inactivation during the first hour of incubation. Salt (NaCl) only had a minor effect on FMDV inactivation. Phosphate supplemented salt treatment increased the effect temperature had on inactivation of CSFV. In contrast, the salt (NaCl) treatment only increased CSFV inactivation at the higher temperatures (20 °C and 25 °C). Also SVDV inactivation was increased by phosphate supplemented salt, but salt (NaCl) treatment only resulted in a significant decrease of SVDV titre at a few time points. The ASFV results showed that both salt (NaCl) and phosphate supplemented salt were capable to inactivate ASFV within 48 h. In contrast to the other viruses (FMDV, CSFV and SVDV), ASFV was the most stable virus even at higher temperatures. The results obtained in this in vitro model underline the efficacy of a combined treatment using phosphate supplemented salt and storage at 20 °C or higher for a period of 30 days. This treatment may therefore be useful in reducing the animal health risks posed by spread of contagious animal viruses by international trade of natural sausage casings.

To establish a reproducible bacterial infection model that mimics the natural route of respiratory tract infection, we developed a computer-controlled nose-only aerosol exposure system for pigs. Actinobacillus pleuropneumoniae aerosols were generated, characterized, and administered dose dependently. Four groups of three unanesthetized pigs were exposed to defined aerosols ranged between 9.2 × 103 to 3.4 × 109 CFU per dose. Graded clinical stages and pathomorphological changes from inapparent carrier state to fatal septicaemia were induced in a dose dependent fashion. Pleuropneumonia in pigs offers a large animal inhalation model to study respiratory tract infections and the origin and function of the local immune system.

The severity of respiratory syncytial virus (RSV) infections appears to differ with age in both humans and bovines. A primary RSV infection in naïve infants and in young calves runs a more severe course when they are 1–6 months old than in their first month of life. The relative lack of clinical signs in the first month of age may be due to high levels of maternally derived neutralizing antibodies or low exposure to infectious virus. This study examined whether age-dependent differences in the pathogenesis of bovine RSV (bRSV) between neonatal and young calves may be due to differences in age-dependent immunocompetence. To study the effect of age and immune parameters on bRSV disease in neonatal and young calves, neonatal (1-day-old) calves without maternally derived antibodies were infected experimentally with bRSV and the severity of disease and immune responses were evaluated in comparison with disease in similar 6-week-old infected calves. Neonatal calves had more extensive virus replication and lung consolidation, but lower pro-inflammatory [in particular tumour necrosis factor alpha (TNF-{alpha})] responses, specific humoral immune responses, lung neutrophilic infiltration and clinical signs of disease than 6-week-old calves. The lack of correlation between virus replication and clinical signs suggests an important role of pro-inflammatory cytokines, in particular TNF-{alpha}, in the disease. The capacity to produce pro-inflammatory TNF-{alpha} appeared to increase with age, and may explain the age-dependent differences in RSV pathogenesis.

Background Worldwide, hepatitis E virus (HEV) genotype 3 is observed in pigs and transmission to humans is implied. To be able to estimate public health risks from e.g. contact with pigs or consumption of pork products, the transmission routes and dynamics of infection should be identified. Hence, the course of HEV-infection in naturally infected pigs should be studied. Results To resemble natural transmission, 24 HEV-susceptible pigs were infected either by one-to-one exposure to intravenously inoculated pigs (C1-pigs; n = 10), by one-to-one exposure to contact-infected pigs (C2-pigs: n = 7; C3-pigs: n = 5) or due to an unknown non-intravenous infection route (one C2-pig and one C3-pig). The course of HEV-infection for contact-infected pigs was characterized by: faecal HEV RNA excretion that started at day 7 (95% confidence interval: 5–10) postexposure and lasted 23 (19–28) days; viremia that started after 13 (8–17) days of faecal HEV RNA excretion and lasted 11 (8–13) days; antibody development that was detected after 13 (10–16) days of faecal HEV RNA excretion. The time until onset of faecal HEV RNA excretion and onset of viremia was significantly shorter for iv-pigs compared to contact-infected pigs, whereas the duration of faecal HEV RNA excretion was significantly longer. At 28 days postinfection HEV RNA was detected less frequently in organs of contact-infected pigs compared to iv-pigs. For contact-infected pigs, HEV RNA was detected in 20 of 39 muscle samples that were proxies for pork at retail and in 4 of 7 urine samples. Conclusion The course of infection differed between infection routes, suggesting that contact-infection could be a better model for natural transmission than iv inoculation. Urine and meat were identified as possible HEV-sources for pig-to-pig and pig-to-human HEV transmission.

Pleuritis in slaughter pigs has increased in recent years in the Netherlands. The aim of the present study was to determine what respiratory pathogens were involved in pleuritis. In total, lungs of 968 slaughter pigs from 10 herds with high prevalence of pleuritis were morphologically examined for size, location, and type of lesions. Moreover, histology and bacteriology were performed. Examination of gross lung lesions showed 45% pleuritis, 14% pleuropneumonia and 38% catarrhal pneumonia. Peribronchiolar cuffing was found in 61 of 142 samples. Actinobacillus pleuropneumoniae was cultured from 22 lung samples from four herds. Pasteurella multocida was cultured from 55 lung samples in eight herds. No specific pattern with respect to the causal pathogens was found. In conclusion, no single infectious cause of pleuritis was found. A variety of infectious agents combined with environmental factors should be considered as a cause of pleuritis.

SIR, — To date, at least three different phylogenetic groups of porcine circo-virus type 2 (pcv-2) have been recognised ([Gagnon and others 2007][1], [Dupont and others 2008][2], [Grau-Roma and others 2008][3], [Timmusk and others 2008][4]). However, the mandate of the International Committee on

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.

Respiratory syncytial virus (RSV) is a major cause of severe respiratory disease in infants and calves. Bovine RSV (bRSV) is a natural pathogen for cattle, and bRSV infection in calves shares many features with the human infection. Thus, bRSV infection in cattle provides the ideal setting to evaluate the safety and efficacy of novel RSV vaccine strategies. Here, we have evaluated the efficacy and safety of modified vaccinia virus Ankara (rMVA)-based vaccine candidates, expressing the bovine RSV-F protein, either or not in combination with the G protein, in colostrums-deprived SPF calves born by caesarean section. Vaccination induced bRSV-specific IgG and CD8 T cell responses. Importantly, no IgE responses were detected. After bRSV challenge, rMVA vaccinated calves experienced less severe symptoms of lower respiratory tract disease compared to the mock-immunized control group. Immunized animals showed reduced pulmonary virus loads, and no eosinophilic infiltration or enhanced respiratory distress. In conclusion, candidate rMVA/bRSV vaccines induced protective and safe immune responses in calves.

Malabsorption syndrome (MAS) in broilers is characterized by enteritis and reduced body weight gain. The pathogenesis of the intestinal lesions and the reasons for susceptibility differences between broiler lines are not clear. We studied the development of enteric lesions, epithelial apoptosis, and cell proliferation in relation to susceptibility. One-day-old chickens from two broiler lines were orally inoculated with intestinal homogenate derived from MAS-affected chickens. Vacuolar degeneration and apoptosis of the villous epithelium and infiltration of heterophils into the lamina propria occurred from day 1 post-inoculation. Following heterophil accumulation, at day 4 to 6 post-inoculation, there was severe apoptosis of the crypt epithelium and villous atrophy. The susceptible broilers had a significantly greater influx of heterophils and, subsequently, severe epithelial apoptosis and cystic damage to the crypts. There appeared to be a causal relationship between heterophil influx and the onset of apoptosis. Coincident with the epithelial apoptosis, MAS-affected chickens had crypt hyperproliferation and faster epithelial turnover. Heterophil infiltration and epithelial apoptosis appear to be critical in the pathogenesis of MAS. Heterophil recruitment may be a major factor in differences in susceptibility to MAS.

Sepsis with subsequent multisystem organ failure after translocation of bacteria from the gut is a serious risk associated with stress situations. We showed that intestinal bacterial translocation could be one of the pathways for pathogenic Streptococcus suis infections in the pig. In 24 piglets weighing 10¿14 kg, free of the extracellular factor (EF+) producing phenotype of S. suis serotype 2, a silicon canula was placed in the proximal jejunum to enable intestinal inoculation and bypassing the upper alimentary tract. The pigs were individually housed. After stress induction in 18 pigs by means of a truck drive in individual cages for 1 h, pigs were inoculated through the intestinal canula either with S. suis type 2 EF+ or with growth medium only, and put back in their original housing. The six not transported pigs were also inoculated with the same strain. To prevent oral self-infection, faeces were collected in a bag that was glued around the anus. Clinical and behavioral symptoms were recorded for 72 h post inoculation, and then the animals were sacrificed for pathological and bacteriological examination. In three animals, the inoculation strain was re-isolated from mesenterial lymph nodes and typically affected organs. No S. suis type 2 EF+ was detected by specific polymerase chain reaction (PCR) in any of the tonsil-swabs and -homogenates. We concluded that infection of the organs had taken place after bacterial translocation out of the gut and that the intestinal tract can be a porte d¿entree for S. suis type 2 EF+.

Type IV pili (Tfp) of gram-negative species share many characteristics, including a common architecture and conserved biogenesis pathway. Much less is known about the regulation of Tfp expression in response to changing environmental conditions. We investigated the diversity of Tfp regulatory systems by searching for the molecular basis of the reported variable expression of the Tfp gene cluster of the pathogen Actinobacillus pleuropneumoniae . Despite the presence of an intact Tfp gene cluster consisting of four genes, apfABCD , no Tfp were formed under standard growth conditions. Sequence analysis of the predicted major subunit protein ApfA showed an atypical alanine residue at position −1 from the prepilin peptidase cleavage site in 42 strains. This alanine deviates from the consensus glycine at this position in Tfp from other species. Yet, cloning of the apfABCD genes under a constitutive promoter in A. pleuropneumoniae resulted in pilin and Tfp assembly. Tfp promoter- luxAB reporter gene fusions demonstrated that the Tfp promoter was intact but tightly regulated. Promoter activity varied with bacterial growth phase and was detected only when bacteria were grown in chemically defined medium. Infection experiments with cultured epithelial cells demonstrated that Tfp promoter activity was upregulated upon adherence of the pathogen to primary cultures of lung epithelial cells. Nonadherent bacteria in the culture supernatant exhibited virtually no promoter activity. A similar upregulation of Tfp promoter activity was observed in vivo during experimental infection of pigs. The host cell contact-induced and in vivo-upregulated Tfp promoter activity in A. pleuropneumoniae adds a new dimension to the diversity of Tfp regulation.

To study adherence of Actinobacillus pleuropneumoniae to porcine lower respiratory epithelium, a cell culture model was developed using primary cultures of porcine lung epithelial cells (LEC). Adherence assays were performed and results were compared with data obtained with swine kidney cells (SK6). A. pleuropneumoniae efficiently adhered to LEC with up to 62 bacteria per cell after 2 h of incubation. Reference strain of serotype 3 (R3) adhered better to LEC than reference strains of serotypes 1 (R1), 7 (R7) and 8 (R8). Overall the adherence to LEC was more rapid and up to 30-fold more efficient than adherence to SK6 cells. In search for the mechanism involved in the adherence event, we tested the effect of LPS which has previously been demonstrated to cause adherence of the pathogen to upper respiratory epithelium. Adherence assays with LPS transposon mutants demonstrated unaltered (mutant with modification in core/lipid A moiety) or even three-fold more adherence (mutants lacking O antigen) compared to the parent micro-organisms. Purified LPS of strains R1, R3, R7 and R8 did not inhibit adherence of R8 to LEC either, suggesting that LPS and particularly the O-antigen are not essential for adherence of A. pleuropneumoniae to LEC. The efficient, LPS-independent adherence of A. pleuropneumoniae to LEC cells indicates that A. pleuropneumoniae may carry different, cell type-specific adhesins and that primary cultures of lower respiratory epithelium are valuable infection models in studying A. pleuropneumoniae pathogenesis.

Most serotypes of A. pleuropneumoniae produce more than one toxin in vivo. To determine the value of the production of more than one toxin in the development of disease, we tested the pathogenicity of isogenic strains of A. pleuropneumoniae serotype 1 that are mutated in the toxin genes apxIA and/or apxIIA or in the transport genes apxIBD. Bacteria mutated in both apxIA and apxIIA, or in apxIBD, were unable to induce pathological lesions, thereby confirming the conclusion that ApxI and ApxII are essential for the pathogenesis of pleuropneumonia. Infection with isogenic strains lacking either ApxI or ApxII did not consistently lead to pleuropneumonia unlike the parent strain S4074. ApxII seemed at least as important as ApxI for the development of clinical and pathological symptoms. Only one of the four pigs inoculated with a mutant strain unable to produce ApxII developed mild pneumonia whereas two out of the three pigs inoculated with a mutant strain unable to produce ApxI developed more severe lesions. The results indicate that both ApxI and ApxII of A. pleuropneumoniae serotype 1 are necessary for full virulence.

The only vaccine currently available to prevent tuberculosis, M. bovis BCG, has limited efficacy. BCG is a poor inducer of CD8+ T cells, which are particularly important for the control of latent tuberculosis and protection against reactivation TB. The induction of strong CD8+ T cell responses is a hallmark of DNA vaccines. We postulated that combining the existing BCG vaccine with plasmid DNA encoding immunodominant antigens of M. tuberculosis, could improve the vaccine by generating stronger CD8+ (and CD4+) T cell responses. As a prototype tuberculosis antigen, the mycolyl-transferase Ag85A was selected. Three groups of six pigs each, were vaccinated intradermally with BCG alone, BCG combined with empty vector or BCG combined with codon-optimized V1j.ns-tPA-Ag85A vector. The BCG-pAg85A DNA combination induced strongly increased and sustained humoral and Th1 type cellular responses against Ag85A and against the purified protein derivative of tuberculin. Increased IFN-γ producing CD8 T cells were detected by intracellular cytokine staining and an Ag85A T cell epitope with predicted MHC class I motif, was promiscuously recognized by 5/6 pigs vaccinated with the combination. In summary, our study provides the first proof of concept in a large mammalian species, for a new TB vaccine, based on a BCG-pDNA combination.

The protective efficacy of a live and killed non-encapsulated isogenic mutant of Streptococcus suis serotype 2 was determined in pigs, and compared with the efficacy of the capsulated wild-type strain. SPF pigs were vaccinated twice intramuscularly at 4 and 7 weeks of age with a dose of 1 x 10(9) formalin-killed CFU of the wild-type (WT-BAC), formalin-killed non-encapsulated mutant (CM-BAC) or live non-encapsulated mutant (CM-LIVE) strain. After 2 weeks, vaccinated pigs and non-vaccinated controls were challenged intravenously with 1 x 10(7) CFU of the homologous, wild-type S. suis serotype 2 strain. Protection was evaluated by clinical, bacteriological, serological and post-mortem examinations. All pigs vaccinated with WT-BAC were completely protected against challenge with the homologous serotype. Pigs vaccinated with CM-BAC were partially protected. Although all pigs vaccinated with CM-BAC survived the challenge, four out of five pigs developed clinical signs of disease for several days. Compared to the WT-BAC and CM-BAC, the CM-LIVE vaccine was less protective. Two out of five pigs vaccinated with CM-LIVE died in the course of the experiment and all of them developed specific clinical signs of disease for several days. The protective efficacy of the vaccines could be associated with serum antibody titers. Antibody titers against cells of wild-type and non-encapsulated mutant strains as well as against muramidase-released proteins (MRP) were high in pigs vaccinated with WT-BAC and CM-BAC. Pigs vaccinated with CM-LIVE showed lower antibody titers. Antibody titers against purified capsular polysaccharides (CPS) of S. suis serotype 2 were only found in pigs vaccinated with WT-BAC. These findings indicate that CPS and other bacterial components of WT-BAC are probably essential for full protection against homologous challenge.

The identification of environmentally regulated genes of Streptococcus suis by the use of iron-restricted conditions in vitro and by experimental infection of piglets is described. Eighteen unique iron-restriction-induced (iri) genes and 22 unique in-vivo-selected (ivs) genes of Strep. suis were found. None of the ivs genes was exclusively expressed in vivo. Four iri genes were identical to four clones selected in piglets. Two ivs genes were similar to genes for putative virulence factors. One of these ivs genes was identical to the epf gene of virulent Strep. suis serotype 2 strains and the other showed homology to a gene encoding a fibronectin-binding protein of Streptococcus gordonii. Two additional ivs genes showed homology to environmentally regulated genes previously identified by using an in vivo expression technology (IVET) selection system in other bacterial species. One of these showed similarity to the agrA gene of Staphylococcus aureus, a key locus involved in the regulation of numerous virulence proteins. The promoter selection system described in this paper has been successfully used for the identification of many environmentally regulated genes potentially involved in the pathogenesis of Strep. suis infections in piglets.

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.

The hypothesis tested in this study was that the reported beneficial effects of spray-dried porcine plasma (SDPP) on piglet post-weaning performance and health are associated with a trophic effect on small intestinal mucosa. At 24 days of age, the piglets of seven sows were assigned to one of three treatment groups. One group continued to be suckled. The other two groups were weaned and offered a diet containing either 15 per cent SDPP or casein. From each treatment group, one piglet was anaesthetised and samples were taken from the small intestinal wall at 26, 28 and 31 days of age. There were no significant effects of SDPP versus casein on villus length. On average, there was less mitotic activity in the SDPP -fed piglets than in those fed casein on days 4 and 7 after weaning. As less mitotic activity leads to less immature enterocytes, this may provide a mechanism for the reported beneficial effects of SDPP on performance and health.

Diseases are often thought to result from a single cause. Although this is sometimes the case, e.g. with a highly virulent infection such as Classical Swine Fever (CSF), more often clinical disease in swine herds results from multiple predisposing factors. This is especially true in modern intensive pig husbandry, in which the role of highly infectious diseases is limited to (nonetheless devastating) outbreaks. More important nowadays are diseases, although associated with an agent, without a clear pathogenesis. The emphasis in disease control thus far has been on treatment, eradication and prevention. This has been achieved by focusing attention on husbandry factors, such as climate, housing, hygiene, management, and nutrition. Although this approach has been successful for a number of diseases, several health problems are persistent. There are strong indications that in the latter, intrinsic animal factors are important. Successful handling of these problems requires knowledge of the (patho)physiology of the pig. In this article, several characteristics of pig physiology associated with the occurrence of disease are described. It appears that the modern (fattening) pig is exceptional among other animal species in that its cardiovascular system is mismatched to its body weight. It is argued that this particular disposition causes relatively minor disturbances to have major consequences in the pig. This concept of pig physiology is central to the understanding of the hitherto poorly understood pathogenesis of several diseases, such as oedema disease.

Streptococcus suis strains (n=411), isolated from diseased pigs in seven European countries were serotyped using specific antisera against serotype 1 to 28, and were phenotyped on the basis of their muramidase-released-protein (MRP) and extracellular-factor protein (EF) production. Overall, S. suis serotype 2 appeared to be most prevalent (32%), followed by serotype 9 (20%) and serotype 1 (12%). Serotype 2 was most frequently isolated in France, Italy and Spain, whereas serotype 9 was most frequently isolated in Belgium, The Netherlands and Germany. In the United Kingdom serotypes 1 and 14 were most frequently isolated. High percentages of S. suis serotype 1, 2, 1/2 and 14 strains, isolated from tissues associated with S. suis infections such as brain, serosa, joint, heart and organs expressed the EF-protein, indicating that in these serotypes expression of EF is likely to be associated with virulence. In contrast, strains belonging to serotype 7 and 9, isolated from tissues associated with S. suis infections did not produce EF. These results strongly suggest that in the serotypes 7 and 9 EF expression is not related to virulence. More than 80% of the S. suis serotype 9 strains produced an MRP* protein, a high molecular variant of the 136kDa MRP. Expression of MRP* in serotype 9 strains is possibly associated with virulence.

Outbreaks of respiratory disease constitute a major health problem in herds of finishing pigs and their aetiology often remains unclear. In this study, 16 outbreaks of respiratory disease with acute clinical signs in finishing pigs were investigated to determine which infectious agents were involved. From each herd four diseased and two clinically healthy pigs were examined pathologically and for the presence of viruses, bacteria and mycoplasmas. In addition, paired blood samples from 10 groupmates of the diseased pigs were tested for antibodies against commonly known causal agents of respiratory disease. A clear diagnosis was possible in 12 of the 16 outbreaks. Seven were due to an infection with influenza virus and five were due to an infection with Actinobadllus pleuropneumoniae . A combination of influenza virus and A pleuropneumoniae may have caused one other outbreak, but no clear cause could be established for the other three outbreaks.

Staphylococcus aureus is one of the most important pathogens of the bovine mammary gland. The interaction of S. aureus with cells of the bovine mammary gland is considered to play an essential role in the pathogenesis. In this study, we identified a new target cell for S. aureus adhesion and invasion. For that purpose, cells which compose the alveoli of the mammary gland were cultured. In these cultures, two morphologically different cell types, elongated and cubic cells, were observed. Adhesion and invasion of S. aureus was studied using microscopical and microbiological methods. S. aureus adhered specifically and in large numbers (about 300 bacteria/cell) to the elongated cell type. No adhesion to the cubic cell type was observed. In addition, bacteria were also found intracellularly in the elongated cells, and enclosed in membrane vesicles. Adhesion and invasion were time dependent and reached maximum levels after 4 h. Invasion was strongly reduced by staurosporine and genistein. The newly identified target cell was further characterized.

We developed a PCR assay for the rapid and sensitive detection of virulent Streptococcus suis type 2 and highly virulent S. suis type 1 in tonsillar specimens from pigs. The PCR primers were based on the sequence of the gene encoding the EF-protein of virulent S. suis type 2 strains (MRP EF ) and highly virulent S. suis type 1 strains (MRP(s)EF ) and of the EF protein of weakly virulent S. suis type 2 strains (MRP EF). The latter strains give rise to larger PCR products than the virulent strains of S. suis type 1 and 2. A positive control template was included in the assay to identify false negative results. The PCR was evaluated using tonsillar specimens from herds known (or suspected) to be infected and herds without an S. suis history. The results obtained with the PCR assay were compared with the results obtained with a newly developed bacteriological examination. In this bacteriological examination we were able to identify the EF-positive strains directly in the tonsillar specimens. From the 99 tonsils examined, 48 were positive in the PCR and 51 negative. All specimens from which EF-positive S. suis strains were isolated were also positive in the PCR assay. Three samples were positive in the PCR, but negative by bacteriological examination. The results demonstrated that the PCR is a highly specific and sensitive diagnostic tool for the detection of pigs carrying virulent strains of S. suis type 2 and highly virulent strains of type 1. Application of the assay may contribute to the control of S. suis infections.

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

Streptococcus suis infections are a common cause of meningitis, septicemia, arthritis, sepsis and sudden death in young pigs and meningitis in humans. During the last few years, S. suis infections have become a major problem in almost all countries with an intensive pig industry. Attempts to control the disease are hampered by the lack of effective vaccines and suitable diagnostics. Until now 32 different serotypes of S. suis have been described, but the serotypes 1 and 2 are isolated most frequently. Within serotypes strains can differ in virulence.

To establish the role of the Apx toxins in the pathogenesis of porcine pleuropneumonia, specific-pathogen-free pigs were inoculated deeply endobronchially with either culture filtrates of Actinobacillus pleuropneumoniae serotype 8 or 9, culture filtrates depleted of the Apx toxins by affinity chromatography, depleted culture filtrate supplemented with purified recombinant Apx toxins (rApx), or purified rApx toxins alone. Results of these experiments indicate that ApxI, ApxIII, and, to a lesser extent, ApxII are the bacterial factors that trigger the development of clinical symptoms and lung lesions typical for porcine pleuropneumonia.

At present only potential virulence factors of Streptococcus suis serotype 2 have been described (2) Reliable laboratory animal models for S. suis infection may aid to identify virulence factors and immunogenic antigens. Since pathogenicity of bacterial species, among which S. suis may not necessarily be the same for mice and pigs, we validated an experimental murine model in which various paramaters (eg microbiological status, route of inoculation, age, breed, and inoculum size had been standardised (1). Therefore we inoculated mice with 13 S. suis type 2 strains, that were selected on their virulence for pigs namely highly virulent for pigs (HV), weakly virulent for pigs (WV) and were nonvirulent for pigs (NV)(7).

In order to outline basic concepts for the design of a bacterial aerosol infection model, the development of a pig model with Actinobacillus pleuropneumoniae is described. First, reproducibility of aerosol parameters should be maintained by optimizing generating and sampling conditions. Survival rates of the chosen strain must be predictable. Secondly, inhalation conditions for the recipients have to be standardized to enable the determination of deposition sites and the dose administered. Subsequently, dose-response relationship should be evaluated to find a suitable challenge dose. Furthermore, it seems necessary to establish methods to obtain local specimens for determination of the local immune responses. The present study demonstrates that after aerosol challenge pigs were completely protected after inhalation and partially protected after oral application of A. pleuropneumoniae vaccines and describes techniques to administer bacteria in a dose-dependent, viable way. Using the infection model several stages of the disease from acute pleuropneumonia to chronic infection can be induced for research purposes.

Strain Henrichsen S 735 (NCTC 10234) of Streptococcus suis serotype 2 reference and three other such strains (strains S 4005, S 3921 and T 141) were tested for virulence by inoculating pigs intranasally and intravenously. The taxonomical properties of each strain were determined. Phenotypes were determined by Western blotting based on MRP and EF protein expression and genotypes were determined by Southern hybridization analysis of the mrp and epf genes. Reference strain S 735 and strain S 3921 produced the 136 kDa MRP and a 180 kDa from of EF, and hence these strains belong to the MRP + EF ∗ phenotype. In accordance with previous experiments with this phenotype, strains S 735 and S 3921 appeared to be only weakly virulent for newborn gnotobiotic pigs. Strain S 4005 produced the 136 kDa MRP and the 110 kDa form of EF, hence it belongs to the MRP + EF + phenotype. This strain was highly virulent for pigs. Strain T 141 did not produce MRP or EF, and hence belongs to the MRP-EF — phenotype. It was nonvirulent for pigs. The route of inoculation did not influence the frequency or severity of clinical signs of disease or lesions, which demonstrated that the 110 kDa EF is not essential during invasion. Southern blot analysis showed that all four S. suis type 2 strains contain sequences that are homologous to the epf and mrp genes. For studies on pathogenesis of S. suis type 2 infections in pigs, we recommend the use of strains that have been tested in a standardized pig model and that belong to the MRP +EF + phenotype, such as strain S 4005.

A dose-defined nose-only inhalation system for pigs was used to study the immunogenic and protective potentials of a single aerosol applicaton of viable or killed Actinobacillus pleuropneumoniae serotype 9. Respiratory volumes were measured for each pig to calculate inhaled individual doses. Eight pigs inhaled 107 CFU A. pleuropneumoniae CVI 13261 reference strain for serotype 9. Another eight pigs received an identical dose of killed actinobacilli. After three weeks the pigs and non-exposed controls were challenged with 108 CFU of the homologous strain by aerosol. Bronchoalveolar lavage (BALF) in pigs was performed during the experiment to obtain lavage samples for assessment of local antibodies. Isotype-specific antibody responses in serum and BAL fluids were measured by ELISAs based on whole-cell antigens. The protective efficacy of aerosol immunization was evaluated by clinical and post-mortem examinations. The controls developed fever and severe pleuropneumonia, whereas previously exposed pigs had less fever and less extensive gross pulmonary lesions. After the first aerosol exposure pulmonary IgM, and IgG antibodies reactive with A. pleuropneumoniae increased signifcantly in both aerosol exposed groups. IgA in BALF and serum concentrations of each Ig class were significantly increased in the group exposed to viable bacteria when compared to the non-exposed controls. After aerosol challenge a pronounced increase of systemic and pulmonary IgA, IgM, and IgG antibodies was detected in both exposure groups. Aerosol application of whole-cell A. pleuropneumoniae bacterins induced similar protective effects against aerosol challenge infection as administration of an identical dose of viable bacteria. Inhalation of A. pleuropnemoniae may lead to asymptomatic carriers in some pigs that could spread the disease under field conditions.

The ribotype profiles of 42 different Streptococcus suis strains were studied. These strains belonged to five serotypes and differed in their virulence for pigs as well as in the expression of the muramidase-released protein and the extracellular protein factor. For the ribotyping, chromosomal DNAs were digested with EcoRI and were hybridized with a 1,066-bp ribosomal DNA probe. The hybridization patterns showed genetic heterogeneity within and between the serotypes. Pathogenic strains of serotype 2 and highly pathogenic strains of serotype 1 could be recognized by their unique ribotype profiles. Nonpathogenic strains showed a high degree of genetic heterogeneity. Moreover, by comparing the 16S ribosomal DNA sequences of a number of S. suis strains, we were able to design two DNA probes which specifically hybridized with S. suis strains.