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Birds can manipulate offspring sex ratio under natural and experimental conditions and maternal hormones have been shown to be involved in this process. Studies also provided evidence for the presence of sex specific concentrations of yolk hormones in avian eggs. These findings led to the suggestion that yolk hormones could influence genetic sex determination in birds. However, in previous studies, yolk hormone concentrations and egg sex were studied in incubated eggs, although incubation of the eggs and embryonic development can alter yolk hormone concentrations and measured sex ratio. This study is the first to determine a wide array of egg components and hen body weight in relation to the sex of the egg in unincubated eggs. Egg parameters studied were yolk concentrations of testosterone, estradiol, androstenedione, progesterone, dihydrotestosterone, and glucose, and egg weight and dimensions. In addition, we studied the associations among all measured parameters. Associations were found between a number of yolk hormones (progesterone associated with testosterone, estradiol and androstenedione; androstenedione with testosterone; dihydrotestosterone with estradiol and androstenedione) as well as between yolk testosterone and egg length and egg weight. There were no significant overall differences between male and female chicken eggs in any of the measured egg parameters. However, there were a few interactions such as the interaction of egg sex with dihydrotestosterone and with hen body weight which predicted estradiol levels and an interaction of estradiol levels with egg width for predicting sex of egg. Their biological relevance need, however, further study. (c) 2013 Elsevier Inc. All rights reserved.

This study investigated the effect of four different nutritional interventions, applied during the first week of life, on immune competence parameters of broiler chickens. The four (dietary) interventions were antibiotics in the drinking water, 25% inclusion of rye in the feed, beta-glucans from Saccharomyces cerevisiae, and coated butyrate. Broiler chickens (between 1 and 2 weeks of age) were subjected to a Necrotic Enteritis (NE) challenge. A positive and negative control treatment (challenged vs. non-challenged chickens, respectively) was included to estimate the effect of the NE challenge. It was hypothesized that applying a nutritional intervention in the first week of life would affect the microbiota colonization, immune system programming, and consequently the quantitative disease phenotype (lesions scores as a result of the NE challenge). The NE challenge did affect the overall performance during the first 2 weeks post-challenge. The antibiotics group showed significant effects on different biological levels: (temporary) increased performance, deviating microbiota composition, increased gene expression in barrier function processes, whereas decreased gene expression in immune related processes, and a higher villi to crypt ratio compared to the (un)challenged control treatment. In the other treatments only significant differences in bacterial genera were observed. In conclusion, this study has shown that it is probable to disturb the gut system development, whereby giving antibiotics (amoxicillin) gives an effect on multiple biological levels. In addition, the other dietary perturbations, i.e. change in feed composition (25% inclusion of rye), feed additives (beta-glucans or coated butyrate) only affected the microbiota composition to some extent.

Het doel van dit project is om een “meetlat” te ontwikkelen die de effecten van (voedings)interventies gericht op de verbetering van de immuuncompetentie van varkens, pluimvee en vleeskalveren kan vaststellen. Immuuncompetentie is binnen dit project gedefinieerd als het vermogen van dieren om effectieve responsen van het immuunsysteem te tonen op het moment dat de gezondheid van het dier onder druk wordt gezet. Een meetlat voor immuuncompetentie kan in de toekomst door de diervoedingssector gebruikt worden bij de ontwikkeling en evaluatie van nieuwe voerconcepten, ingrediënten en additieven gericht op de verbetering en ondersteuning van diergezondheid. Het is bekend dat de samenstelling van de voeding van jonge dieren invloed heeft op de functionele ontwikkeling van het maagdarmkanaal en op de samenstelling van de daarin aanwezige microbiota. De interacties tussen de microbiota en de weefsels van het darmkanaal (cross talk) hebben een belangrijke invloed op de ontwikkeling van immuuncompetentie. Daarom wordt in dit project gefocust op de effecten van (voedings)interventies op de microbiota, genexpressie veranderingen in darmweefsel, en morfologische en immunologische veranderen in de darm. De hier gepresenteerde meetlat voor immuuncompetentie is gebaseerd op de resultaten van onderzoek binnen het VDI programma van Feed4Foodure (projecten VDI-11; vleeskuikens, VDI-12; biggen, VDI- 13; gespeende biggen en kalveren) waarin m.b.v. model interventies de effecten van variatie in voersamenstelling op de microbiota samenstelling in het darmkanaal, de biologische responsen van darmweefsel en de zoötechnische dierprestaties zijn onderzocht. In de hier gepresenteerde meetlat worden gemeten effecten in deze studies aan elkaar gerelateerd en functioneel inzichtelijk gemaakt. Dit rapport beschrijft de ontwikkeling en totstandkoming van een eerste versie van de meetlat. Hierbij worden gemaakte keuzes, beperkingen en mogelijkheden van de meetlat bediscussieerd. Tenslotte wordt inzicht gegeven in de mogelijkheden tot verdere verfijningen en de toepasbaarheid van de meetlat.

Background Gene expression profiles of intestinal mucosa of chickens and pigs fed over long-term periods (days/weeks) with a diet rich in rye and a diet supplemented with zinc, respectively, or of chickens after a one-day amoxicillin treatment of chickens, were recorded recently. Such dietary interventions are frequently used to modulate animal performance or therapeutically for monogastric livestock. In this study, changes in gene expression induced by these three interventions in cultured “Intestinal Porcine Epithelial Cells” (IPEC-J2) recorded after a short-term period of 2 and 6 hours, were compared to the in vivo gene expression profiles in order to evaluate the capability of this in vitro bioassay in predicting in vivo responses. Methods Lists of response genes were analysed with bioinformatics programs to identify common biological pathways induced in vivo as well as in vitro. Furthermore, overlapping genes and pathways were evaluated for possible involvement in the biological processes induced in vivo by datamining and consulting literature. Results For all three interventions, only a limited number of identical genes and a few common biological processes/pathways were found to be affected by the respective interventions. However, several enterocyte-specific regulatory and secreted effector proteins that responded in vitro could be related to processes regulated in vivo, i.e. processes related to mineral absorption, (epithelial) cell adherence and tight junction formation for zinc, microtubule and cytoskeleton integrity for amoxicillin, and cell-cycle progression and mucus production for rye. Conclusions Short-term gene expression responses to dietary interventions as measured in the in vitro bioassay have a low predictability for long-term responses as measured in the intestinal mucosa in vivo. The short-term responses of a set regulatory and effector genes, as measured in this bioassay, however, provided additional insight into how specific processes in piglets and broilers may be modulated by “early” signalling molecules produced by enterocytes. The relevance of this set of regulatory/effector genes and cognate biological processes for zinc deficiency and supplementation, gluten allergy (rye), and amoxicillin administration in humans is discussed. Electronic supplementary material The online version of this article (doi:10.1186/s12263-017-0559-1) contains supplementary material, which is available to authorized users.

A novel orthobunyavirus, named "Schmallenberg virus" (SBV), was first detected in the blood of cattle at the end of the summer in Germany in 2011, and subsequently in late autumn from the brain of a stillborn malformed lamb in The Netherlands. Full genome sequences, including 5' and 3' terminal "panhandle" sequences of the L, M, and S segments of the SBV isolated from lamb brain tissue (named HL1) were determined. In addition, a second SBV strain was isolated from the blood of a dairy cow (named F6) also in The Netherlands. This isolate was passaged on Vero cells, and its genome sequence was determined by next-generation sequencing. Alignments of the two genome sequences revealed 4, 12, and 2 amino acid differences in the open reading frames of the L, M, and S segments, respectively. Eleven of a total of 12 amino acid differences were detected in the M segment encoding the ectodomain of the putative structural glycoprotein Gc. Notably, in the HL1 isolate, positions 737-739 are occupied by isoleucine, arginine, and leucine (IRL), whereas in the majority of other sequenced SBV isolates these positions are occupied by threonine, histidine, and proline, respectively. Moreover, in all sheep, goat, and cattle SBV isolates sequenced and published so far, an IRL sequence was never found. This has brought us to the conclusion that the M segment of the HL1 isolate differed markedly from that of other lamb and cow isolates. Whether this atypical variant resulted from adaptation to the ewe, fetus, or insect vector remains to be investigated.

Infection of pigs with CSFV can lead to either acute disease, resulting in death or recovery, or chronic disease. The mechanisms by which CSFV manipulates the pig’s first line of defence to establish a chronic infection are poorly understood. Therefore, pigs were infected with moderately virulent CSFV, and whole blood was collected on a regular basis during a period of 18 days. Using whole-genome microarrays, time-dependent changes in gene expression were recorded in blood cells of chronically diseased pigs and pigs that recovered. Bioinformatics analysis of regulated genes indicated that different immunological pathways were regulated in chronically diseased pigs compared to recovered pigs. In recovered pigs, antiviral defence mechanisms were rapidly activated, whereas in chronically diseased pigs, several genes with the potential to inhibit NF-¿B- and IRF3/7-mediated transcription of type I interferons were up-regulated. Compared to recovered pigs, chronically diseased pigs failed to activate NK or cytotoxic T-cell pathways, and they showed decreased gene activity in antigen-presenting monocytes/macrophages. Remarkably, in chronically diseased pigs, genes related to the human autoimmune disease systemic lupus erythematosus (SLE) were up-regulated during the whole period of 18 days. CSFV pathology in kidney and skin resembles that of SLE. Furthermore, enzymes involved in the degradation of 1,25-dihydroxyvitamin D3 and of tryptophan to kynurenines were expressed at different levels in chronically diseased and recovered pigs. Both of these chemical processes may affect the functions of T helper/regulatory cells that are crucial for tempering the inflammatory response after a viral infection.

Worldwide infectious diarrhoea, mainly caused by rotavirus and enterotoxigenic Escherichia coli (ETEC), accounts for a large part of deaths in children. ETEC is also the main cause of traveller's diarrhoea. Probiotics are promising for prevention and treatment of diarrhoea, but there is insufficient evidence to support the use of any specific probiotic or probiotics in general. Because of the sensitivity of suckling and weaned piglets for ETEC, piglets are a good model for infectious diarrhoea in infants and traveller's diarrhoea. Just as in human the efficacy of probiotics in diminishing diarrhoea and improving growth in suckling and weaned piglets is not uniform. A piglet model of infectious diarrhoea provides access to intestinal compartments that are not easily accessible in infants. In an in situ piglet model of secretory diarrhoea, the functional physiological response to ETEC and the concomitant host genome response to ETEC and probiotics may be tested. This will provide new insights in the complex crosstalk between ETEC, probiotics and the gut in the future.

The immune response to CSFV and the strategies of this virus to evade and suppress the pigs' immune system are still poorly understood. Therefore, we investigated the transcriptional response in the tonsils, median retropharyngeal lymph node (MRLN), and spleen of pigs infected with CSFV strains of similar origin with high, moderate, and low virulence. Using a porcine spleen/intestinal cDNA microarray, expression levels in RNA pools prepared from infected tissue at 3 dpi (three pigs per virus strain) were compared to levels in pools prepared from uninfected homologue tissues (nine pigs). A total of 44 genes were found to be differentially expressed. The genes were functionally clustered in six groups: innate and adaptive immune response, interferon-regulated genes, apoptosis, ubiquitin-mediated proteolysis, oxidative phosphorylation and cytoskeleton. Significant up-regulation of three IFN-gamma-induced genes in the MRLNs of pigs infected with the low virulence strain was the only clear qualitative difference in gene expression observed between the strains with high, moderate and low virulence. Real-time PCR analysis of four response genes in all individual samples largely confirmed the microarray data at 3 dpi. Additional PCR analysis of infected tonsil, MRLN, and spleen samples collected at 7 and 10 dpi indicated that the strong induction of expression of the antiviral response genes chemokine CXCL10 and 2'-5' oligoadenylate synthetase 2, and of the TNF-related apoptosis-inducing ligand (TRAIL) gene at 3 dpi, decreased to lower levels at 7 and 10 dpi. For the highly and moderately virulent strains, this decrease in antiviral and apoptotic gene expression coincided with higher levels of virus in these immune tissues.

Host-microorganism interactions in the intestinal tract are complex, and little is known about specific nonpathogenic microbial factors triggering host responses in the gut. In this study, mannose-specific interactions of Lactobacillus plantarum 299v with jejunal epithelium were investigated using an in situ pig Small Intestinal Segment Perfusion model. The effects of L. plantarum 299v wild-type strain were compared with those of two corresponding mutant strains either lacking the gene encoding for the mannose-specific adhesin (msa) or sortase (srtA; responsible for anchoring of cell surface proteins like Msa to the cell wall). A slight enrichment of the wild-type strain associated with the intestinal surface could be observed after 8 h of perfusion when a mixture of wild-type and msa-mutant strain had been applied. In contrast to the mutant strains, the L. plantarum wild-type strain tended to induce a decrease in jejunal net fluid absorption compared with control conditions. Furthermore, after 8 h of perfusion expression of the host gene encoding pancreatitis-associated protein, a protein with proposed bactericidal properties, was found to be upregulated by the wild-type strain only. These observations suggest a role of Msa in the induction of host responses in the pig intestine.

Germ-free piglets were orally infected with virulent rotavirus to collect jejunal mucosal scrapings at 12 and 18 hours post infection (two piglets per time point). IFN-gamma mRNA expression was stimulated in the mucosa of all four infected piglets, indicating that they all responded to the rotavirus infection. RNA pools prepared from two infected piglets were used to compare whole mucosal gene expression at 12 and 18 hpi to expression in uninfected germ-free piglets (n = 3) using a porcine intestinal cDNA microarray. Microarray analysis identified 13 down-regulated and 17 up-regulated genes. Northern blot analysis of a selected group of genes confirmed the data of the microarray. Genes were functionally clustered in interferon-regulated genes, proliferation/differentiation genes, apoptosis genes, cytoskeleton genes, signal transduction genes, and enterocyte digestive, absorptive, and transport genes. Down-regulation of the transport gene cluster reflected in part the loss of rotavirus-infected enterocytes from the villous tips. Data mining suggested that several genes were regulated in lower- or mid-villus immature enterocytes and goblet cells, probably to support repair of the damaged epithelial cell layer at the villous tips. Furthermore, up-regulation was observed for IFN-γ induced guanylate binding protein 2, a protein that effectively inhibited VSV and EMCV replication in vitro (Arch Virol 150:1213–1220, 2005). This protein may play a role in the small intestine’s innate defense against enteric viruses like rotavirus. Electronic supplementary material The online version of this article (doi:10.1007/s00705-008-0118-6) contains supplementary material, which is available to authorized users.

The pestivirus glycoprotein E(rns), a ribonuclease, is expressed on the surface of virions and in infected cells as a disulfide-linked homodimer. E(rns) is involved in the infection process and its RNase activity is probably involved in viral replication and pathogenesis. The most C-terminal cysteine residue forms an intermolecular disulfide bond with another E(rns) monomer, resulting in an E(rns) dimer. To study the function of dimerisation of E(rns) for viral replication, the cysteine residue at amino acid position 438 was mutated into a serine residue. The mutated C438S gene was cloned into a vector containing an infectious cDNA copy of the CSFV C-strain genome. Using reverse genetics, a mutant virus was generated that only expressed monomeric E(rns), confirming that Cys 438 is essential for homo-dimerization. Characterization of this mutant virus and of a baculovirus-expressed C438S mutant protein indicated that the loss of the dimeric state of E(rns) reduced the affinity of binding of virions and E(rns) to heparan sulphate (HS), the receptor for E(rns) on the cell surface of SK6 cells. This suggests that interaction of virus-bound E(rns) homodimers with membrane associated HS may be a joined action of the two HS-binding domains (one in each monomer) present in the homodimer.

To study host–probiotic interactions in parts of the intestine only accessible in humans by surgery (jejunum, ileum and colon), pigs were used as model for humans. Groups of eight 6-week-old pigs were repeatedly orally administered with 5 × 1012 CFU Lactobacillus plantarum 299v (L. plantarum 299v) or PBS, starting with a single dose followed by three consecutive daily dosings 10 days later. Gene expression was assessed with pooled RNA samples isolated from jejunum, ileum and colon scrapings of the eight pigs per group using Affymetrix porcine microarrays. Comparison of gene expression profiles recorded from L. plantarum 299v-treated pigs with PBS-treated pigs indicated that L. plantarum 299v affected metabolic and immunological processes, particularly in the ileum. A higher expression level of several B cell-specific transcription factors/regulators was observed, suggesting that an influx of B cells from the periphery to the ileum and/or the proliferation of progenitor B cells to IgA-committed plasma cells in the Peyer’s patches of the ileum was stimulated. Genes coding for enzymes that metabolize leukotriene B4, 1,25-dihydroxyvitamin D3 and steroids were regulated in the ileum. Bioinformatics analysis predicted that these metabolites may play a role in the crosstalk between intestinal immune cells and sub-mucosal adipocytes. Together with regulation of genes that repress NFKB- and PPARG-mediated transcription, this crosstalk may contribute to tempering of inflammatory reactions. Furthermore, the enzyme adenosine deaminase, responsible for the breakdown of the anti-inflammatory mediator adenosine, was strongly down-regulated in response to L. plantarum 299v. This suggested that L. plantarum 299v-regulated production of adenosine by immune cells like regulatory T cells may also be a mechanism that tempers inflammation in the ileum, and perhaps also in other parts of the pig’s body. Electronic supplementary material The online version of this article (doi:10.1007/s12263-015-0461-7) contains supplementary material, which is available to authorized users.

Passage of native classical swine fever virus (CSFV) in cultured swine kidney cells (SK6 cells) selects virus variants that attach to the surface of cells by interaction with membrane-associated heparan sulfate (HS). A Ser-to-Arg change in the C terminus of envelope glycoprotein E rns (amino acid 476 in the open reading frame of CSFV) is responsible for selection of these HS-binding virus variants (M. M. Hulst, H. G. P. van Gennip, and R. J. M. Moormann, J. Virol. 74:9553–9561, 2000). In this investigation we studied the role of binding of CSFV to HS in vivo. Using reverse genetics, an HS-independent recombinant virus (S-ST virus) with Ser 476 and an HS-dependent recombinant virus (S-RT virus) with Arg 476 were constructed. Animal experiments indicated that this adaptive Ser-to-Arg mutation had no effect on the virulence of CSFV. Analysis of viruses reisolated from pigs infected with these recombinant viruses indicated that replication in vivo introduced no mutations in the genes of the envelope proteins E rns , E1, and E2. However, the blood of one of the three pigs infected with the S-RT virus contained also a low level of virus particles that, when grown under a methylcellulose overlay, produced relative large plaques, characteristic of an HS-independent virus. Sequence analysis of such a large-plaque phenotype showed that Arg 476 was mutated back to Ser 476 . Removal of HS from the cell surface and addition of heparin to the medium inhibited infection of cultured (SK6) and primary swine kidney cells with S-ST virus reisolated from pigs by about 70% whereas infection with the administered S-ST recombinant virus produced in SK6 cells was not affected. Furthermore, E rns S-ST protein, produced in insect cells, could bind to immobilized heparin and to HS chains on the surface of SK6 cells. These results indicated that S-ST virus generated in pigs is able to infect cells by an HS-dependent mechanism. Binding of concanavalin A (ConA) to virus particles stimulated the infection of SK6 cells with S-ST virus produced in these cells by 12-fold; in contrast, ConA stimulated infection with S-ST virus generated in pigs no more than 3-fold. This suggests that the surface properties of S-ST virus reisolated from pigs are distinct from those of S-ST virus produced in cell culture. We postulate that due to these surface properties, in vivo-generated CSFV is able to infect cells by an HS-dependent mechanism. Infection studies with the HS-dependent S-RT virus, however, indicated that interaction with HS did not mediate infection of lung macrophages, indicating that alternative receptors are also involved in the attachment of CSFV to cells.

Linseed as a feed ingredient contains a number of constituents, such as mucilage, gel forming polysaccharides, structural carbohydrates and ω-3 fatty acids, which could have functional properties in relation to maintaining and supporting gastro-intestinal health in post weaning piglets. Intestinal gene expression was studied in weanling piglets fed diets containing either or not linseed or linseed expeller meal. Piglets were either or not orally challenged with an enterotoxigenic strain of E. coli K88. Pairwise comparisons identified several mRNAs expressed significantly different, in particular between treatments C− (control, not challenged) and LS− (linseed, not challenged) and LS+ (linseed, challenged) and LSM+ (linseed expeller meal, challenged). The intestinal micro-array technique is a promising tool to study the relationship between nutrition and feeding and gut tissue responses under defined conditions.

Bovine foamy virus (BFV) infections are highly prevalent among cattle worldwide. However, relatively little is known about the impact of this virus on the host immune system. In our study, we focused on a bovine macrophage cell line (BoMac) and examined changes in the BoMac transcriptome after in vitro infection with BFV using bovine BLOPlus oligo microarrays. One hundred twenty-four genes showed significant changes in expression level. The biological process categories found to be enriched include metabolic processes, cell communication, transport, immune system processes, and response to extracellular stimuli. RT-qPCR was applied to confirm the results obtained for representative genes.

Pure preparations of envelope glycoproteins E(rns) and E2 of classical swine fever virus (CSFV) synthesized in insect cells were used to study infection of porcine and bovine cells with the pestiviruses CSFV and bovine viral diarrhoea virus (BVDV). Almost 100% inhibition of infection of porcine kidney cells with CSFV was produced by 100 microg/ml E(rns). After removal of the virus no E(rns) was needed in the overlay medium (growth medium) to maintain this level of inhibition. In contrast, 100% inhibition of infection of porcine kidney cells with CSFV by 10 microg/ml E2 was only achieved when E2 was added to the overlay medium. When E2 was omitted, a maximum of 50% inhibition was achieved. This indicated that after the virus and E2 were removed from the cells, infection still occurred, by virus particles which were still bound to the cell surface. Treatment with 100 microg/ml E(rns) released these particles from the cell surface. Furthermore, E(rns) bound irreversibly to the surface of cells susceptible or unsusceptible to pestivirus infection and cell-to-cell spread of CSFV was completely inhibited by E2 but not by E(rns). These results demonstrated that E(rns) and E2 interacted with different cell surface receptors. Inhibition of BVDV infection of porcine and bovine cells by CSFV E2 suggested that CSFV E2 and BVDV E2 share an identical receptor. BVDV strain 5250 isolated from pigs was efficiently inhibited by CSFV E(rns), whereas several BVDV strains isolated from cattle were not, suggesting that the conformation of E(rns) plays a role in host tropism.

Classical swine fever virus (CSFV; synonym hog cholera virus), bovine viral diarrhoea virus (BVDV), and border disease virus (BDV), members of the Pestivirus genus of the family of Flaviviridae (Francki et al., 1991) are small, enveloped, positive-stranded RNA viruses (Moennig & Plagemann, 1992). The viruses are structurally, antigenically and genetically closely related. BVDV and BDV can infect ruminants and pigs. CSFV infections are restricted to pigs. The envelope of the CSF virion contains three glycoproteins, El (gp51 to gp54), E2 (gp42 to gp46), and E3 (gp31) (Moormann et al., 1990; Thiel et al., 1991). Animals infected with pestiviruses raise antibodies against at least two viral glycoproteins, namely El and E2 (Kwang et al., 1992; Terpstra & Wensvoort, 1988). El is the most immunodominant protein present in the envelope and plays an important role in virus neutralization (Wensvoort et al., 1990). CSFV causes a highly contagious and often fatal disease in pigs. Outbreaks of the disease occur intermittently in several European countries and can cause large economical losses. An active serological screening program followed by “stamping out” of infected herds, is used to control outbreaks of the disease. This policy is not only expensive but is also not sufficient for eradication of classical swine fever in Europe. Vaccination of pigs with a live attenuated CSFV vaccine strain, the C strain, protects pigs from classical swine fever

In birds, offspring sex ratio manipulation by mothers is now well established with potentially important consequences for evolution and animal breeding. In most studies on primary sex ratio of birds, eggs are sexed after incubation by the use of PCR methods targeted to the sex-linked CHD1 genes. Sexing of unincubated eggs would be preferred, but as fertile and infertile blastodiscs cannot be distinguished macroscopically, errors could arise from PCR amplifications of parental DNA associated with the vitelline membrane of infertile eggs. In this study, we stained blastodiscs without the vitelline membrane with Hoechst 33342. This allowed unequivocal distinction between fertile and infertile blastodiscs. Fertile blastodiscs contained thousands of fluorescent nuclei, whereas no nuclei were seen in infertile eggs. In addition, after nucleic acid analysis, fertile blastodiscs yielded much stronger chromosomal DNA and CHD1-targeted PCR bands on agarose gels compared with infertile blastodiscs. These findings indicate that fertile blastodiscs contain much more embryonic DNA than parental DNA, allowing reliable sexing of the fertile eggs. The differences between fertile and infertile blastodiscs in chromosomal DNA and CHD1 PCR banding intensities alone could also be used to distinguish fertile from infertile eggs without using Hoechst staining. We conclude that identifying fertile blastodiscs either by Hoechst staining or by analyzing the yield of chromosomal DNA and CHD1-PCR products, combined with CHD1-targeted PCR amplification, presents an easy and reliable method to sex unincubated eggs.

The processing and protective capacity of E1, an envelope glycoprotein of hog cholera virus (HCV), were investigated after expression of different versions of the protein in insect cells by using a baculovirus vector. Recombinant virus BacE1[+] expressed E1, including its C-terminal transmembrane region (TMR), and generated a protein which was similar in size (51 to 54 kDa) to the size of E1 expressed in swine kidney cells infected with HCV. The protein was not secreted from the insect cells, and like wild-type E1, it remained sensitive to endo-beta-N-acetyl-D-glucosaminidase H (endo H). This indicates that E1 with a TMR accumulates in the endoplasmic reticulum or cis-Golgi region of the cell. In contrast, recombinant virus BacE1[-], which expressed E1 without a C-terminal TMR, generated a protein that was secreted from the cells. The fraction of this protein that was found to be cell associated had a slightly lower molecular mass (49 to 52 kDa) than wild-type E1 and remained endo H sensitive. The high-mannose units of the secreted protein were trimmed during transport through the exocytotic pathway to endo H-resistant glycans, resulting in a protein with a lower molecular mass (46 to 48 kDa). Secreted E1 accumulated in the medium to about 30 micrograms/10(6) cells. This amount was about 3-fold higher than that of cell-associated E1 in BacE1[-] and 10-fold higher than that of cell-associated E1 in BacE1[+]-infected Sf21 cells. Intramuscular vaccination of pigs with immunoaffinity-purified E1 in a double water-oil emulsion elicited high titers of neutralizing antibodies between 2 and 4 weeks after vaccination at the lowest dose tested (20 micrograms). The vaccinated pigs were completely protected against intranasal challenge with 100 50% lethal doses of HCV strain Brescia, indicating that E1 expressed in insect cells is an excellent candidate for development of a new, safe, and effective HCV subunit vaccine.

The genome of Lelystad virus (LV), the causative agent of porcine epidemic abortion and respiratory syndrome (previously known as mystery swine disease), was shown to be a polyadenylated RNA molecule. The nucleotide sequence of the LV genome was determined from a set of overlapping cDNA clones. A consecutive sequence of 15,088 nucleotides was obtained. Eight open reading frames (ORFs) that might encode virus-specific proteins were identified. ORF1a and ORF1b are predicted to encode the vital RNA polymerase because the amino acid sequence contains sequence elements that are conserved in RNA polymerases of the torovirus Berne virus (BEV), equine arteritis virus (EAV), lactate dehydrogenase-elevating virus (LDV), the coronaviruses, and other positive-strand RNA viruses. A heptanucleotide slippery sequence (UUUAAAC) and a putative pseudoknot structure, which are both required for efficient ribosomal frameshifting during translation of the RNA polymerase ORF 1b of BEV, EAV, and the coronaviruses, were identified in the overlapping region of ORF1a and ORF1b of LV. ORFs 2 to 6 probably encode viral membrane-associated proteins, whereas ORF7 is predicted to encode the nucleocapsid protein. Comparison of the amino acid sequences of the ORFs identified in the genome of LV, LDV, and EAV indicated that LV and LDV are more closely related than LV and EAV. A 3′ nested set of six subgenomic RNAs was detected in LV-infected cells. These subgenomic RNAs contain a common leader sequence that is derived from the 5′ end of the genomic RNA and that is joined to the 3′ terminal body sequence. Our results indicate that LV is closely related evolutionarily to LDV and EAV, both members of a recently proposed family of positive-strand RNA viruses, the Arteriviridae.

The present study aimed to investigate whether long-lasting, recurrent tethering of sows leads to enduring effects on measures that may be indicative of chronic stress. Sows that had experienced tethering for about 1.5 or 4.5 years and age-matched sows kept in a social housing system (loose sows) were compared. Immediately after slaughter, blood samples were taken to measure plasma cortisol levels, and the brain, spleen, and adrenals were dissected and weighed. Gene expression in the frontal cortex and hippocampus was analyzed. Plasma cortisol levels were higher in the tethered sows than in the loose sows. The older, but not the younger, tethered sows had heavier adrenal glands than their loose counterparts. The weight of the spleen was not affected by the housing conditions, but the pituitary gland was lighter in tethered sows than in loose sows. Microarray analyses revealed an increased expression of beta-globin mRNA in the hippocampus and to a lesser extent in the frontal cortex of the older tethered sows, compared with the older loose sows. Taken together, the findings indicate that chronically stressed pigs develop depression-like symptoms. However, it can be questioned whether the pig subjected to repeated, long-term stress can be regarded an animal model of major depression.

Acute secretory diarrhea is a major cause of morbidity and mortality in young animals and humans. Deaths result from excessive fluid and electrolyte losses. The disease is caused by non-invasive bacteria such as Vibrio cholerae and Escherichia coli which produce enterotoxins, however, much less is known about the role of individual host responses. Here we report the response of intact porcine small intestinal mucosa to infection with enterotoxigenic E. coli (ETEC). Jejunal segments in four piglets were infused with or without ETEC, and perfused for 8 h, and net absorption measured. Microarray analysis at 8 h post-infection showed significant differential regulation of on average fifteen transcripts in mucosa, with considerable individual variation. Differential net absorption varied between animals, and correlated negatively with the number of up regulated genes, and with one individual gene (THO complex 4). This shows that quantitative differences in gene regulation can be functionally linked to the physiological response in these four animals.

This paper reviews the laboratory investigations that led us to isolate the Lelystad virus and demonstrate that this virus causes mystery swine disease. We describe: 1) isolating the virus from the disease; 2) characterizing the virus as a new enveloped RNA virus; 3) reproducing the disease experimentally with the isolated Lelystad virus; 4) isolating the virus from the experimentally induced disease.

Genomic RNA of hog cholera virus (HCV) strain Brescia was cloned and sequenced. The nucleotide sequence was deduced from overlapping cDNA clones and comprises 12,283 nucleotides. We cloned the complete 3′ end of the HCV genome, but could not unequivocally prove that the cDNA sequence also completely covers HCV RNA at the 5′ end. The HCV genome contained one large open reading frame, which spans the viral plus strand RNA and encodes an amino acid sequence of 3898 residues with a calculated molecular weight of 438,300. To identify structural HCV glycoproteins, we prepared rabbit antisera against three synthetic peptides deduced from the sequence. Because one of these antisera reacted with a 51- to 54-kDa glycoprotein (envelope protein Et of HCV) on Western blot, the genomic position of the sequence encoding gp51–54 could be clearly established. The amino acid sequence of Brescia was compared with that of HCV strain Alfort and that of BVDV strains NADL and Osloss. The degree of homology between the two HCV strains was 93%, and between Brescia and the BVDV strains about 70%. NADL contained an inserted sequence of 90 amino acids that was absent from the sequences of Brescia, Alfort, and Osloss, whereas Osloss contained an inserted sequence of 76 amino acids that was absent from the sequences of Brescia, Alfort, and NADL. Sequences in p80, the most homologous protein among pestiviruses, showed similarity to six sequence motifs found conserved in helicase-like proteins represented by elF-4A. Furthermore, a trypsin-like serine protease domain detected in p80 of BVDV was also found conserved in HCV, suggesting that pestivirus p80 may be bifunctional.

The intestine is a complex and dynamic ecosystem, in which nutrients, exogenous compounds and micro-flora interact, and its condition is influenced by the complex interaction between these factors and host genetic elements. Furthermore, interactions of immune cells with the other components of the intestinal mucosa are essential in the defense against pathogens. The outcomes of these complex interactions determine resistance to infectious diseases. The development of genomic tools and techniques allows for analysis of multiple and complex host responses. We have constructed a porcine small intestinal micro-array, based on cDNA from jejunal mucosal scrapings. Material from two developmental distinct stages (4- and 12-week-old pigs) was used in order to assure a reasonably broad representation of mucosal transcripts. The micro-array consists of 3468 cDNAs spotted in quadruplicate. Comparison of the 4-week-old versus 12-week-old pigs revealed a differential expression in at least 300 spots. Furthermore, we report the early gene expression response of pig small intestine jejunal mucosa to infection with enterotoxigenic E. coli (ETEC) using the small intestinal segment perfusion (SISP) technique. A response pattern was found in which a marker for innate defense dominated, demonstrating the strength of this applied technology. Further analysis of these response patterns will contribute to a better understanding of enteric health and disease in pigs. The great similarity between pig and human suggest results from these continuing studies should be applicable for both agricultural and human biomedical purposes.

Two related classical swine fever virus (CSFV) strain Brescia clones were isolated from blood samples from an infected pig. Virus C1.1.1 is a cell-adapted avirulent variant, whereas CoBrB is a virulent variant. Sequence analysis revealed 29 nucleic acid mutations in C1.1.1, resulting in 9 amino acid substitutions compared to the sequence of CoBrB 476 R. Using reverse genetics, parts of the genomes of these viruses, which contain differences that lead to amino acid changes, were exchanged. Animal experiments with chimeric viruses derived from C1.1.1 and CoBrB 476 R showed that a combination of amino acid changes in the structural and nonstructural regions reduced the virulence of CSFV in pigs. Moreover, the presence of a Leu at position 710 in structural envelope protein E2 seemed to be an important factor in the virulence of the virus. Changing the Leu at position 710 in the CoBrB 476 S variant into a His residue did not affect virulence. However, the 710 His in the C1.1.1/CoBrB virus, together with adaptive mutations 276 R, 476 R, and 477 I in E rns , resulted in reduced virulence in pigs. These results indicated that mutations in E rns and E2 alone do not determine virulence in pigs. The results of in vitro experiments suggested that a high affinity for heparan sulfate of C1.1.1 E rns may reduce the spread of the C1.1.1/CoBrB virus in pigs and together with the altered surface structure of E2 caused by the 710 L→H mutation may result in a less efficient infection of specific target cells in pigs. Both these features contributed to the attenuation of the C1.1.1/CoBrB virus in vivo.

Publisher Summary Together with flaviviruses and hepatitis C virus (HCV), an important human pathogen, the pestiviruses are classified as a genus within the family Flaviviridae. Viruses in this family are small, enveloped, positive-strand RNA viruses. Compared with HCV, the pestivirus genome encodes two additional proteins, an N-terminal autoprotease, Npro, and the envelope protein Erns. Comparison of the amino acid sequences of Erns of pestiviruses with amino acid sequences in databases identified two short stretches, both eight amino acids in length, that are homologous to the active site domains of ribonucleases of the RNase T2 family. Enzymatic tests of purified Erns proved that these stretches are involved in ribonuclease activity. This chapter reviews the specific properties of this RNase activity and its function in relation to the life cycle of pestiviruses. The properties of the RNase activity of CSFV Erns were determined with purified native Erns, and Erns purified from insect cells. For ribonucleases of the RNase T2 family, it has been shown that the histidine residues in the two conserved domains are essential for RNase catalysis. Mutational studies showed that this is also true for Erns. Besides the possible cytotoxic action of Erns toward the host immune system there is also a possible role for Erns RNase activity in regulation of RNA synthesis in infected cells.

Infection of cells with Classical swine fever virus (CSFV) is mediated by the interaction of envelope glycoprotein E rns and E2 with the cell surface. In this report we studied the role of the cell surface glycoaminoglycans (GAGs), chondroitin sulfates A, B, and C (CS-A, -B, and -C), and heparan sulfate (HS) in the initial binding of CSFV strain Brescia to cells. Removal of HS from the surface of swine kidney cells (SK6) by heparinase I treatment almost completely abolished infection of these cells with virus that was extensively passaged in swine kidney cells before it was cloned (clone C1.1.1). Infection with C1.1.1 was inhibited completely by heparin (a GAG chemically related to HS but sulfated to a higher extent) and by dextran sulfate (an artificial highly sulfated polysaccharide), whereas HS and CS-A, -B, and -C were unable to inhibit infection. Bound C1.1.1 virus particles were released from the cell surface by treatment with heparin. Furthermore, C1.1.1 virus particles and CSFV E rns purified from insect cells bound to immobilized heparin, whereas purified CSFV E2 did not. These results indicate that initial binding of this virus clone is accomplished by the interaction of E rns with cell surface HS. In contrast, infection of SK6 cells with virus clones isolated from the blood of an infected pig and minimally passaged in SK6 cells was not affected by heparinase I treatment of cells and the addition of heparin to the medium. However, after one additional round of amplification in SK6 cells, infection with these virus clones was affected by heparinase I treatment and heparin. Sequence analysis of the E rns genes of these virus clones before and after amplification in SK6 cells showed that passage in SK6 cells resulted in a change of an Ser residue to an Arg residue in the C terminus of E rns (amino acid 476 in the polyprotein of CSFV). Replacement of the E rns gene of an infectious DNA copy of C1.1.1 with the E rns genes of these virus variants proved that acquisition of this Arg was sufficient to alter an HS-independent virus to a virus that uses HS as an E rns receptor.

Envelope glycoprotein E rns of classical swine fever virus (CSFV) has been shown to contain RNase activity and is involved in virus infection. Two short regions of amino acids in the sequence of E rns are responsible for RNase activity. In both regions, histidine residues appear to be essential for catalysis. They were replaced by lysine residues to inactivate the RNase activity. The mutated sequence of E rns was inserted into the p10 locus of a baculovirus vector and expressed in insect cells. Compared to intact E rns , the mutated proteins had lost their RNase activity. The mutated proteins reacted with E rns -specific neutralizing monoclonal and polyclonal antibodies and were still able to inhibit infection of swine kidney cells (SK6) with CSFV, but at a concentration higher than that measured for intact E rns . This result indicated that the conformation of the mutated proteins was not severely affected by the inactivation. To study the effect of these mutations on virus infection and replication, a CSFV mutant with an inactivated E rns (FLc13) was generated with an infectious DNA copy of CSFV strain C. The mutant virus showed the same growth kinetics as the parent virus in cell culture. However, in contrast to the parent virus, the RNase-negative virus induced a cytopathic effect in swine kidney cells. This effect could be neutralized by rescue of the inactivated E rns gene and by neutralizing polyclonal antibodies directed against E rns , indicating that this effect was an inherent property of the RNase-negative virus. Analyses of cellular DNA of swine kidney cells showed that the RNase-negative CSFV induced apoptosis. We conclude that the RNase activity of envelope protein E rns plays an important role in the replication of pestiviruses and speculate that this RNase activity might be responsible for the persistence of these viruses in their natural host.

Classical swine fever virus and bovine virus diarrhea virus are members of the genus pestivirus, which belongs to the family of the Flaviviridae. Recently, envelope glycoprotein Erns was identified as an RNase. RNases can express different biological actions. They have been shown to be neurotoxic, antihelminthic, and immunosuppressive. We studied the immunosuppressive properties of Erns in vitro. The glycoprotein totally inhibited concanavalin A-induced proliferation of porcine, bovine, ovine, and human lymphocytes. We then studied the direct cytotoxic effects of Erns on lymphocytes and epithelial cells in protein synthesis assays. Erns strongly inhibited the protein synthesis of lymphocytes of different species, without cell membrane damage. This suggested an apoptotic process, and indeed apoptosis of lymphocytes was detected after incubation with Erns. Pestivirus infections are characterized by leukopenia and immunosuppression. Our results suggest that Erns plays an important role in the pathogenesis of pestiviruses.

Envelope glycoprotein D (gD) of pseudorabies virus (PRV) is essential for penetration but is not required for cell-to-cell spread. When animals are inoculated with a phenotypically complemented PRV gD mutant, the virus is able to spread locally by means of direct cell-to-cell transmission, but progeny virions released by infected cells are non-infectious because they lack gD. Therefore, the virus cannot be transmitted from inoculated animals to other animals. This property makes a PRV gD mutant an attractive candidate as a safe vaccine vector. To examine whether a self-restricted, non-transmissible PRV mutant can be used as a biologically safe vaccine vector, a gD/gE-negative PRV recombinant virus which expresses envelope glycoprotein E2 of classical swine fever virus was constructed. Vaccination of pigs showed that the recombinant virus was able to protect pigs against both Aujeszky's disease and classical swine fever.

Infectious RNA was transcribed for the first time from a full-length cDNA template of the plus-strand RNA genome of a pestivirus. The genome of the C strain, which is a vaccine strain of classical swine fever virus, was sequenced and used to synthesize the template. The cDNA sequence of the C strain was found to be 12,311 nucleotides in length and contained one large open reading frame encoding a polyprotein of 3,898 amino acids. Although there were mostly only small differences between the sequence of the C strain and the published sequences of strains Alfort and Brescia, there was one notable insertion of 13 nucleotides, TTTTCTTTTTTTT, in the 3' noncoding region of the C strain. Furthermore, we showed that the sequences at the 5' and 3' termini of the C strain are highly conserved among pestiviruses. We found that the infectivity of the in vitro transcripts of DNA copies pPRKflc-113 and pPRKflc-133 depended on the correctness of the nucleotide sequence. The in vitro transcripts of pPRKflc-133 were infectious, whereas those of pPRKflc-113 were not. In fact, only 5 amino acids among the complete amino acid sequence determined this difference in infectivity. However, virus FLc-133, which was generated from pPRKflc-133, cannot be differentiated from native C-strain virus. Therefore, we exchanged the region encoding the antigenic N-terminal half of envelope protein E2 in pPRKflc-133 with the equivalent region of strain Brescia. The resulting hybrid virus, FLc-h6, could be differentiated from the C strain and from FLc-133 with monoclonal antibodies directed against envelope proteins Erns and E2 of strain Brescia and the C strain. To be suitable for further vaccine development, viruses generated from pPRKflc-133 should grow at least as well as native C-strain virus. In fact, we found that FLc-133, hybrid virus FLc-h6, and the C strain grew equally well. We concluded that pPRKflc-133 is an excellent tool for developing a classical swine fever marker vaccine and may prove valuable for studying the replication, virulence, cell and host tropism, and pathogenesis of classical swine fever virus.

Background The aim of this study was to identify transcription factors/regulators that play a crucial role in steering the (innate) immune response shortly (within a few hours) after the first contact of the intestinal mucosa with an inflammatory mediator, and to test whether the processes regulated by these factors/regulators can be modulated by chemical substances of natural origin. Methods We experimentally induced inflammation by perfusion of surgically applied jejunal loops with Salmonella enterica subspecies enterica serovar Typhimurium DT104 in three pigs. Segments of mock and Salmonella treated loops were dissected after 2, 4 and 8¿hours of perfusion. IL8 and IL1-beta mRNA expression levels were measured in mucosal scrapings of all segments. Furthermore, intra-animal microarray comparisons (isogenic) between Salmonella and mock treated segments after 8¿hours, and inter-animal comparisons between similar Salmonella-treated loops of each pig at 2 and 4¿hours, were performed. Results IL-1beta and IL8 mRNA levels, and intra-animal microarray comparisons at 8¿hours between Salmonella and mock treated segments showed that the response-time and type of response to Salmonella was different in all three pigs. This plasticity allowed us to extract a comprehensive set of differentially expressed genes from inter-animal comparisons at 2 and 4¿hours. Pathway analysis indicated that many of these genes play a role in induction and/or tempering the inflammatory response in the intestine. Among them a set of transcription factors/regulators known to be involved in regulation of inflammation, but also factors/regulators for which involvement was not expected. Nine out of twenty compounds of natural origin, which according to literature had the potential to modulate the activity of these factors/regulators, were able to stimulate or inhibit a Salmonella-induced mRNA response of inflammatory-reporter genes IL8 and/or nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha in cultured intestinal porcine epithelial cells. Conclusions We describe a set of transcription factors/regulators possibly involved in regulation of “very early” immune mechanism which determines the inflammatory status of the intestine later on. In addition, we show that these mechanisms may be modulated by chemical substances of natural origin. Keywords: Transcription regulation; Salmonella-induced inflammation; Pig intestine

Brain-derived neurotrophic factor (BDNF) signaling has been implicated in the onset of depression and in antidepressant efficacy, although the exact role of this neurotrophin in the pathophysiology of depression remains to be elucidated. Also, the interaction between chronic stress, which may precede depression, corticosteroids and BDNF is not fully understood. The present study aimed at investigating whether long-lasting, recurrent tethering of sows during a period of 1.5 or 4.5 years leads to enduring effects on measures that may be indicative of chronic stress, compared with animals kept in a group housing system ('loose' sows). Immediately after slaughter, the frontal cortex, dorsal and ventral hippocampus were dissected and protein levels of BDNF and its receptors were analyzed and compared with plasma cortisol levels and adrenal weights. Results indicate that tethering stress reduced BDNF protein levels in the dorsal hippocampus and the frontal cortex, but not in the ventral hippocampus. In addition, levels of TrkB, the high affinity receptor for BDNF, were increased in the dorsal hippocampus. Plasma cortisol levels and adrenal weight were increased after tethering. These stress effects on BDNF levels were more pronounced after 4.5 years of recurrent tethering and negatively correlated in particular in the frontal cortex with cortisol levels and adrenal weight. This suggests that the stress effect of tethered housing on neurotrophin levels may be mediated via cortisol. Taken together, these data indicate that recurrent tethering stress in sows over 4.5 years results in a loss of neurotrophic support by BDNF, mediated by an overactive neuroendocrine system.

Two regions of amino acids homologous to the ribonuclease catalysis domain of the fungal RNases T2 of Aspergillus oryzae and Rh of Rhizopus niveus and the plant S-glycoproteins of Nicotiana alata are perfectly conserved in the amino acid sequence of the envelope glycoprotein E2 of classical swine fever virus (CSFV). To analyze the functional significance of these conserved sequences, the gene encoding E2 was inserted into the p10 locus of baculovirus and expressed in insect cells. Recombinant virus BacCE2 generated a protein which was similar in size (42 to 46 kDa) to wild-type E2 synthesized in swine kidney cells infected with CSFV. Recombinant E2 was purified by immunoaffinity chromatography from the lysate of cells infected with BacCE2 and assayed for RNase activity. RNase activity coeluted with the E2 fraction, indicating that ribonuclease activity is an inherent property of E2. The ribonuclease-specific activity of the protein fraction containing pure E2 was comparable to that of the N. alata S-glycoproteins.

Lelystad virus (LV) is an enveloped positive-stranded RNA virus, which causes abortions and respiratory disease in pigs. The complete nucleotide sequence of the genome of LV has been determined. This sequence is 15.1 kb in length and contains a poly(A) tail at the 3′ end. Open reading frames that might encode the viral replicases (ORFs 1a and 1b), membrane-associated proteins (ORFs 2 to 6) and the nucleocapsid protein (ORF7) have been identified. Sequence comparisons have indicated that LV is distantly related to the coronaviruses and toroviruses and closely related to lactate dehydrogenase-elevating virus (LDV) and equine arteritis virus (EAV). A 3′ nested set of six sub- genomic RNAs is produced in LV-infected alveolar lung macrophages. These subgenomic RNAs contain a leader sequence, which is derived from the 5′ end of the viral genome. Altogether, these data show that LV is closely related evolutionarily to LDV and EAV, both members of a recently proposed family of positive-stranded RNA viruses, the Arteriviridae.

To investigate whether live attenuated pseudorabies virus (PRV) can be used as a vaccine vector, PRV recombinants that expressed envelope glycoprotein E1 of hog cholera virus (HCV) were generated. Pigs inoculated with these recombinants developed high levels of neutralizing antibodies against PRV and HCV and were protected against both pseudorabies and hog cholera (classical swine fever).

Monoclonal antibodies (MCAs) against porcine immunoglobulin isotypes G, G 1 , G 2 , M and A have been produced and characterized in detail. Epitope analysis using a competitive direct enzyme-linked immunosorbent assay (ELISA) indicated that the MCAs recognized 3 class-specific epitopes of IgG, 4 epitopes specific for IgG 1 , 3 epitopes specific for IgG 2 , 2 epitopes of IgM and 2 epitopes of IgA. Two MCAs against IgG 2 were shown to react with an allotypic determinant (B2) and one MCA against IgM is probably allotype specific. The production of MCAs specific for IgG and for its subclasses G 1 and G 2 and, in addition, the one-step isolation of nearly pure IgG 1 and IgG 2 preparations by immunoaffinity chromatography using MCA 34.1.1a (anti-IgG 2 ) confirmed the existence of at least two subclasses of IgG. Preliminary results further suggested the existence of a subpopulation of IgG 1 which could be eluted selectively from Protein A-Sepharose columns at pH 5.0. MCA 34.17.2a appeared to react preferentially with this IgG 1 subpopulation and could be used to isolate a similar IgG 1 subpopulation by immuno-affinity chromatography. Several of the MCAs have been successfully applied for the detection of porcine immunoglobulin isotypes by a double antibody sandwich ELISA and for the (isotype-specific) detection of antibodies against various porcine viruses. The availability of a full set of MCAs against porcine immunoglobulin isotypes will stimulate and facilitate the further study of the porcine immune system.

Virus-specific RNA in swine kidney cells infected with hog cholera virus and the RNA of gradient-purified virions were identified and characterized. The time curve for virus replication in SK-6 cells was established. The logarithmic phase of virus growth was between 4 and 12 h after virus adsorption. Virus production was maximum at 20 h after virus adsorption. Analysis of cytoplasmic RNA in a neutral agarose gel showed that the time curve for synthesis of a high molecular weight RNA in infected cells closely paralleled that for virus growth. Electrophoresis in an agarose-formaldehyde gel of [3H]uridine-labeled RNA, synthesized with actinomycin-D, showed that this high molecular weight RNA species was approximately 12 kilobases (kb) in length. RNA, extracted from virions purified in a glycerol-tartrate gradient, co-migrated in a neutral agarose gel with the 12 kb RNA species; moreover, it hybridized specifically with a cDNA clone prepared against the intracellular 12 kb RNA. These results confirm the virus-specific nature of the 12 kb RNA. Since no prominent subgenomic virus-specific RNA was identified in infected cells, an RNA species of this size may also act as a messenger RNA.

Background At the end of 2011, a new orthobunyavirus, tentatively named Schmallenberg virus (SBV), was discovered in Germany. This virus has since been associated with clinical signs of decreased milk production, watery diarrhoea and fever in dairy cows, and subsequently also with congenital malformations in calves, lambs and goat kids. In affected countries, initial surveillance for the infection was based on examination of malformed progeny. These suspicions were followed up by real-time reverse transcription polymerase chain reaction (RT-PCR) on brain tissue. For epidemiological purposes, a serological assay was, however, needed. Results A virus neutralisation test (VNT) was developed and optimized, and subsequently evaluated. This VNT has a specificity of >99% and the sensitivity is likely also very close to 100%. The assay is highly repeatable and reproducible. The final assay was used to test for antibodies in cows, ewes and does from herds known to be infected or suspected to be so. Targets for sampling in these herds were the mothers of malformed offspring. In herds with an RT-PCR confirmed SBV infection, more than 94% (190 out of 201) of the ewes and 99% (145 out of 146) of the cows were seropositive. In herds with suspicion of SBV infection based on birth of malformed offspring only (no or negative RT-PCR), more than 90% (231 out of 255) of the ewes and 95% (795 out of 834) of the cows were seropositive. In goats, on the other hand, only a low number of seropositives was found: overall 36.4%, being 16 out of 44 goats tested. Conclusions Given the characteristics of this VNT, it can be used at a relative high throughput for testing of animals for export, surveillance, screening and research purposes, but can also be used as a confirmation test for commercially available enzyme-linked immunosorbent assays (ELISA’s) and for (relative) quantification of antibodies. Suspicions of SBV infections that were confirmed by RT-PCR were almost always confirmed by serology in cows. Due to individual registration and identification of cows and calves, affected offspring could almost always be traced back to the mother. Ewes on the other hand were not always the mothers of affected lambs, but were in many cases herd mates with unaffected lambs. This indicated a high within-herd seroprevalence of antibodies against SBV.