[Classical swine fever in Germany, in hindsight].
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Results for “Classical Swine Fever”
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Serodiagnostic comparison between two methods, ELISA and surface plasmon resonance for the detection of antibodies of classical swine fever.
A protein chip based on surface plasmon resonance (SPR) was developed to measure the antibody (Ab) titers of classical swine fever virus (CSFV) using the recombinant gp55 protein as an antigen. The diagnostic potential of this SPR assay for detecting the Ab titers to CSFV gp55 was compared that of the enzyme-linked immunosorbent assay (ELISA) using 170 serum samples from 14 pig farms. The SPR assay was highly specific and sensitive, and there were no cross-reactions detected. There was a strong positive correlation between the SPR and ELISA titers (n=170, r=0.869, p<0.01). Therefore, the SPR label-free method is a valuable tool in the serodiagnosis of CSFV infection and determining Ab titers after vaccination.
Animals↗
Oronasal vaccination with classical swine fever virus (CSFV) replicon particles with either partial or complete deletion of the E2 gene induces partial protection against lethal challenge with highly virulent CSFV.
A cDNA clone of the classical swine fever virus (CSFV) strain Alfort/187 [Ruggli N, Tratschin JD, Mittelholzer C, Hofmann MA. Nucleotide sequence of classical swine fever virus strain Alfort/187 and transcription of infectious RNA from stably cloned full-length cDNA. J Virol 1996;70(6):3478-87] was used to construct two E2 deletion mutants lacking either the complete E2 gene or, alternatively, a stretch of 204 nucleotides encoding 68 amino acids located in the C-terminal region of the E2 glycoprotein. The respective in vitro synthesized mutant RNAs replicated in SK-6 cells but no infectious virus was generated. Both replicons could be packaged into virus particles in SK-6 cells constitutively expressing E2 of CSFV. For the resulting CSF virus replicon particles (CSF-VRP) A187-E2del373 and A187-E2del68 titers of 10(6) and 10(7) TCID(50)/ml, respectively, were obtained. Oronasal vaccination with 10(7) TCID(50) of either of the two CSF-VRP protected pigs against a challenge with a lethal dose of CSFV strain Eystrup. In contrast, after intradermal vaccination VRP A187-E2del68 but not VRP A187-E2del373 lacking the complete E2 gene induced a protective immune response. We conclude that E2-complemented CSF-VRP have the potential to be used as live-attenuated non-transmissible oral vaccines for pigs. In addition, our data suggest that E2 of CSFV is dispensable for the induction of mucosal but not of parenteral immunity.
Administration, Intranasal↗
Detection of classical swine fever virus in the ovaries of experimentally infected sows.
Six sows were infected intranasally with a Korean isolate of classical swine fever virus (CSFV). The distribution of virus in ovarian tissues was then assessed for 21 days by in-situ hybridization and immunohistochemistry. CSFV was detected in the ovaries between 7 and 21 days post-inoculation (dpi) by both methods, but the labelling was particularly intense and widespread at 7 dpi. CSFV nucleic acid and antigen were located almost exclusively within the cytoplasm of cells shown by haematoxylin and eosin staining to be macrophages, which were numerous in atretic follicles. Small numbers of CSFV nucleic acid-positive cells with distinctly round morphology and oval nuclei, resembling monocytes, were also observed in the blood vessels of sows at 7 and 14 dpi. CSFV nucleic acid and antigen were not observed in primordial, primary or secondary follicles from infected sows at 7, 14 or 21 dpi. The results suggest that CSFV replicates in circulating peripheral monocytes and gains access to ovarian tissues from the bloodstream, and that this contributes to the distribution of CSFV in macrophages throughout the atretic follicles.
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A histopathologic, immunohistochemical, and ultrastructural study of the intestine in pigs inoculated with classical swine fever virus.
The aim of this study was to report on the lesions occurring in the intestine during experimental classical swine fever (CSF) and to clarify the nature of infected cells and the distribution of viral antigen. Thirty-two pigs were inoculated with the virulent CSF virus (CSFV) isolate Alfort 187 and slaughtered from 2 to 15 postinoculation days; four animals of similar background served as a control group. Immunohistochemistry, electron microscopy, and the transferase-mediated deoxyuridine triphosphate nick-end labeling method were used to detect viral antigens and apoptosis. The results showed progressive lymphoid depletion and mucosal necrosis. The lymphoid depletion could have been caused by apoptosis of lymphocytes but could not be directly attributed to the effect of CSFV on these cells. Vascular changes, pathogenic bacteria, and viral infection of epithelial cells were ruled out as causes of necrotic lesions. However, large virally infected monocytes-macrophages with ultrastructural changes indicative of activation were observed in the intestine. This suggests that monocytes-macrophages play an important role in the pathogenesis of intestinal lesions. An understanding of the function of these cells will require additional study.
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Oral immunisation of wild boar against classical swine fever: concluding analysis of the recent field trials in Germany.
The recent oral immunisation trials in wild boar against classical swine fever (CSF) in Germany are described and evaluated in summary. After the first field study in Lower Saxony from 1993-1995 further immunisation trials started in Mecklenburg-Western Pomerania, Brandenburg, Lower Saxony, Baden-Württemberg and Saxony-Anhalt. The immunisation strategies and the size of the vaccination zones were different in the individual federal states. In principle, the bait vaccine based on the CSF virus strain "C" were laid out by hand. Later also the aerial distribution was carried out in selected areas of Mecklenburg-Western Pomerania. The application of baits by plane was introduced at the beginning of the immunisation measures in Saxony-Anhalt apart from the manual distribution. Up to now, the field trials show that the oral immunisation can be an additional tool for CSF control by increasing of herd immunity and reduction of the CSFV prevalence. However, the immunisation was not sufficient enough for young boars in the most field studies. Based on the evaluation of the immunisation experiments an improved immunisation procedure is recommended.
Administration, Oral↗
Descriptive epidemiology of a classical swine fever outbreak in the Limburg Province of Belgium in 1997.
This paper describes the epidemiological characteristics of the 1997 Classical Swine Fever (CSF) outbreak that occurred in the Limburg Province of Belgium, where there is a policy of non-vaccination, intensive surveillance and eradication. Between 30 June and 17 July 1997, eight herds, located in three different areas, were confirmed to be CSF-positive. CSF virus was transmitted from the primary infected herd of one area to another five herds in the same area and to one herd in a different area. The mode of virus introduction for this primary infected herd and for the one herd that was not infected by this primary herd could not be determined. Clinical, serological, and virological findings indicated that the CSF-infected herds were detected in an early stage of the infection. The early detection of the infection together with a preventive stamping out procedure resulted in a rapid elimination of the CSF virus. A total of 46,561 pigs were slaughtered to control the spread of the infection. Another 27,579 pigs were slaughtered in the framework of the market support. The total direct costs of the episode were estimated at [symbol: see text] 10,893,337.
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Studies on the virulence of two field isolates of the classical Swine Fever virus genotype 2.3 rostock in wild boars of different age groups.
The virulence of two isolates of the classical swine fever virus (CSFV) was studied in experimentally infected wild boars of different ages. The isolates, originating from wild boars shot in Mecklenburg-Western Pomerania (isolate '1829-NVP') and in Rhineland-Palatinate (isolate '11722-WIL'), belong to the genetic subgroup 2.3 Rostock. Clinical picture, transient viraemia, virus excretion and gross lesions at necropsy as well as a failure of virus detection at the end of the experiment revealed that this virus subtype was only moderately virulent. Whereas one subadult wild boar and both 7-week-old wild boar piglets infected intranasally became sick and died, only one of three 8-week-old animals which survived after contact infection remained CSFV positive until the end of the experiment [34 days post infection (dpi)], although neutralizing antibodies were present. This underlines the role of young boars in CSF epidemics. The isolate '11722-WIL' was shed by an infected adult wild boar and was transmitted to susceptible piglets. Interestingly, all animals which became sick and died also were found to be infected with a secondary pathogen. Therefore, we assume that after infection with moderately virulent CSFV simultaneous infections with other pathogens may be important for the clinical course and the outcome of the disease as well as for a spread of the virus in field.
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Comparison of reverse transcriptase-polymerase chain reaction, virus isolation, and immunoperoxidase assays for detecting pigs infected with low, moderate, and high virulent strains of classical swine fever virus.
Pigs were experimentally inoculated with Glentorf, Lelystad/97, and Alfort/187: representative low, moderate, and high virulent strains of classical swine fever virus (CSFV). Animals were tested for viremia using virus isolation and reverse transcriptase-polymerase chain reaction (RT-PCR) assays run under routine diagnostic conditions. The virus was detected in the peripheral blood by virus isolation and RT-PCR assays of all Glentorf- and Lelystad/97-infected pigs beginning at 3 days postinoculation (dpi) and in all Alfort/187-infected pigs beginning at 2 dpi. Viremia, as determined by virus isolation, remained detectable in Lelystad/97- and Alfort/187-infected pigs until the last animal within each cohort was euthanized on days 12 and 7 postinoculation, respectively. In contrast, the virus could be isolated from the blood of all Glentorf-infected pigs between 3 and 7 dpi but not from 10 to 21 dpi when the experiment was concluded. Viremia, as determined by RT-PCR, became apparent in Alfort/187-infected pigs at 2 dpi and in Glentorf- and Lelystad/97-infected pigs at 3 dpi. All pigs, regardless of the CSFV strain used, remained RT-PCR positive until they were euthanized. Tonsils were harvested from all the pigs and frozen sections tested for the presence of the CSFV antigen using polyclonal pestivirus and monoclonal CSFV horseradish peroxidase (HRPO) conjugates. Immunostaining reactions were positive for all the Alfort/187- and Lelystad/97-infected pigs. By contrast, tonsils from the Glentorf-infected pigs gave negative to equivocal results. These data suggest that an RT-PCR assay performed on blood may be the best test when dealing with pigs infected with low virulent strains of CSFV.
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Chimeric (marker) C-strain viruses induce clinical protection against virulent classical swine fever virus (CSFV) and reduce transmission of CSFV between vaccinated pigs.
Two live recombinant vaccines (Flc9 and Flc11) against classical swine fever (CSF) were evaluated for their capacity to reduce transmission of virulent CSF virus (CSFV) among vaccinated pigs. In Flc9 the 5' terminal half of the E2 gene of the C-strain, a CSFV vaccine strain, was exchanged with the homologous gene of the bovine viral diarrhoea virus (BVDV) strain 5250, the E(rns) gene was exchanged likewise in the chimeric Flc11 virus. Both recombinant vaccines induce an antibody response in pigs that can be distinguished from that induced after a wild-type CSFV infection. Four experiments were performed to estimate the reproduction ratio R after different vaccination-challenge intervals. Each group consisted of ten pigs [specified pathogen free (SPF) pigs or conventional pigs] that were vaccinated once, intramuscularly, either with Flc9 or Flc11 virus or that were not vaccinated. Vaccinated and susceptible pigs were challenged intranasally with the virulent CSFV strain Brescia or Behring, 1, 2 or 4 weeks after vaccination. Whether contact-pigs became infected was determined using a CSFV specific E2 (Flc9) or E(rns) (FLc11) antibody ELISA. In the unvaccinated control groups, virus secretion started from day 2 to 4 after inoculation and all contact pigs became infected. Contact pigs became infected in the group of pigs (SPF or conventional) vaccinated once with Flc9 virus and challenged 1-, 2- or 4-weeks later. The estimates of the R in the groups challenged at 1-, 2- and 4-weeks after vaccination were 0.38, 0 and 0.75, respectively. Contact infected pigs were not detected (R=0) in any of the groups of pigs, vaccinated with Flc11, only SPF pigs were used. In order to achieve a statistical significance of R within the vaccinated groups each of the experiments has to be repeated at least once. The R of pigs vaccinated with Flc11 virus and challenged at 1- or 2-weeks after vaccination was however significantly lower that the reproduction ratio of the unvaccinated groups (P=0.013). The R of pigs vaccinated with Flc9 virus and challenged at 1 (conventional pigs) or 2 weeks (SPF pigs) after vaccination was significantly lower that the reproduction ratio of the unvaccinated groups (P=0.013). In conclusion, both chimeric viruses Flc9 and Flc11 provided good clinical protection against a challenge with virulent CSFV at 1 or 2 weeks after vaccination. Further experiments should be carried out to study more aspects of the efficacy of these recombinant viruses before they can be used as a marker vaccine under field circumstances.
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Genetic variation in the 5' end and NS5B regions of classical swine fever virus genome among Japanese isolates.
Sixteen clinical strains of classical swine fever virus (CSFV) isolated in Japan were subjected to analyses of nucleotide sequence variations in the 5' end and NS5B regions of the genome. These isolates were divided into three genovars, CSFV-1, CSFV-2 and CSFV-3, based on palindromic nucleotide substitutions at the three variable loci in the 5' untranslated region (UTR). Phylogenetic trees constructed from nucleotide sequences in the 5'-UTR and NS5B gene indicated that the CSFV strains were divided into three clusters, I, II and III. CSFV strains included in clusters I, II and III were identical to those in the CSFV-1, CSFV-2 and CSFV-3 genovars, respectively.
5' Untranslated Regions↗
Identification of T-cell epitopes in the structural and non-structural proteins of classical swine fever virus.
To identify new T-cell epitopes of classical swine fever virus (CSFV), 573 overlapping, synthetic pentadecapeptides spanning 82% of the CSFV (strain Glentorf) genome sequence were synthesized and screened. In proliferation assays, 26 peptides distributed throughout the CSFV viral protein sequences were able to induce specific T-cell responses in PBMCs from a CSFV-Glentorf-infected d/d haplotype pig. Of these 26 peptides, 18 were also recognized by PBMCs from a CSFV-Alfort/187-infected d/d haplotype pig. In further experiments, it could be shown that peptide 290 (KHKVRNEVMVHWFDD), which corresponds to amino acid residues 1446-1460 of the CSFV non-structural protein NS2-3 could induce interferon-gamma secretion after secondary in vitro restimulation. The major histocompatibility complex (MHC) restriction for stimulation of T-cells by this pentadecapeptide was identified as being mainly MHC class II and partially MHC class I. In cytolytic assays, CSFV-specific cytotoxic T-lymphocytes (CTLs) were able to lyse peptide 290-loaded target cells. These findings indicate the existence of a CSFV-specific helper T-cell epitope and a CTL epitope in this peptide.
Amino Acid Sequence↗
[Identification and comparison of neutralizing epitopes of glycoprotein E(rns) of classical swine fever virus].
Structural and envelope glycoprotein E(rns) (gp48) of classical swine fever virus (CSFV) is the second antigenic protein being responsible for eliciting neutralizing antibodies and conferring protective immunity. Infection of cells with CSFV is mediated by the interaction of glycoprotein E(rns) and E2 with the cell surface receptors. The glycoprotein E(rns) has been shown to contain RNase activity, which plays a role in the viral life cycle and is also involved in virus neutralization. Neutralizing epitopes of glycoprotein E(rns) had been mapped by screening a 12-mer random peptide phage display library using the neutralizing monoclonal antibodies (MAbs) 1B5, b4-22 and 24/16, raised against CSFV strain alfort T bingen and reacted with glycoprotein E(rns). Three major epitope (mimotope) motifs, WxNxxP, DKNR (Q) G and A (T) CxYxKN (around amino acid position aa351-aa356 or aa348-aa350, aa384-aa386 and aa322-aa323, aa380-aa386 of glycoprotein E(rns) of CSFV) were identified respectively and characterized immunologically by the MAbs, 1b5, b4-22 and 24/16. MAbs b4-22 and 24/16 shared a part of binding motif sequence KN, and recognized the similar antigenic domain on the glycoprotein E(rns) but showed a distinct pattern of flank sequence and reactivities with the mimotopes by Western blot and inhibition of immunofluorescent antibody analysis.
Amino Acid Sequence↗
Cost-effectiveness of measures to prevent classical swine fever introduction into The Netherlands.
Recent history has demonstrated that classical swine fever (CSF) epidemics can incur high economic losses, especially for exporting countries that have densely populated pig areas and apply a strategy of non-vaccination, such as The Netherlands. Introduction of CSF virus (CSFV) remains a continuing threat to the pig production sector in The Netherlands. Reducing the annual probability of CSFV introduction (P(CSFV)) by preventive measures is therefore of utmost importance. The choice of preventive measures depends not only on the achieved reduction of the annual P(CSFV), but also on the expenditures required for implementing these measures. The objective of this study was to explore the cost-effectiveness of tactical measures aimed at the prevention of CSFV introduction into The Netherlands. For this purpose for each measure (i) model calculations were performed with a scenario tree model for CSFV introduction and (ii) its annual cost was estimated. The cost-effectiveness was then determined as the reduction of the annual P(CSFV) achieved by each preventive measure (DeltaP) divided by the annual cost of implementing that measure (DeltaC). The measures analysed reduce the P(CSFV) caused by import or export of pigs. Results showed that separation of national and international transport of pigs is the most cost-effective measure, especially when risk aversion is assumed. Although testing piglets and breeding pigs by a quick and reliable PCR also had a high cost-effectiveness ratio, this measure is not attractive due to the high cost per pig imported. Besides, implementing such a measure is not allowed under current EU law, as it is trade restrictive.
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Specific interaction of eukaryotic translation initiation factor 3 with the 5' nontranslated regions of hepatitis C virus and classical swine fever virus RNAs.
Translation of hepatitis C virus (HCV) and classical swine fever virus (CSFV) RNAs is initiated by cap-independent attachment (internal entry) of ribosomes to the approximately 350-nucleotide internal ribosomal entry segment (IRES) at the 5' end of both RNAs. Eukaryotic initiation factor 3 (eIF3) binds specifically to HCV and CSFV IRESs and plays an essential role in the initiation process on them. Here we report the results of chemical and enzymatic footprinting analyses of binary eIF3-IRES complexes, which have been used to identify the eIF3 binding sites on HCV and CSFV IRESs. eIF3 protected an internal bulge in the apical stem IIIb of domain III of the CSFV IRES from chemical modification and protected bonds in and adjacent to this bulge from cleavage by RNases ONE and V1. eIF3 protected an analagous region in domain III of the HCV IRES from cleavage by these enzymes. These results are consistent with the results of primer extension analyses and were supported by observations that deletion of stem-loop IIIb or of the adjacent hairpin IIIc from the HCV IRES abrogated the binding of eIF3 to this RNA. This is the first report that eIF3 is able to bind a eukaryotic mRNA in a sequence- or structure-specific manner. UV cross-linking of eIF3 to [32P]UTP-labelled HCV and CSFV IRES elements resulted in strong labelling of 4 (p170, p116, p66, and p47) of the 10 subunits of eIF3, 1 or more of which are likely to be determinants of these interactions. In the cytoplasm, eIF3 is stoichiometrically associated with free 40S ribosomal subunits. The results presented here are consistent with a model in which binding of these two translation components to separate, specific sites on both HCV and CSFV IRESs enhances the efficiency and accuracy of binding of these RNAs to 40S subunits in an orientation that promotes entry of the initiation codon into the ribosomal P site.
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The correlation of virus-specific interferon-gamma production and protection against classical swine fever virus infection.
The level of antigen-specific interferon-gamma (IFN-gamma) production can be used as an indicator of cellular immunity. In this study, we investigated the role of cellular immune response in protection against classical swine fever virus (CSFV). Pigs were vaccinated once with CSFV vaccine and challenged 6 days post-vaccination (dpv). Vaccinated animals had significantly higher CSFV-specific IFN-gamma secreting cells than the unvaccinated pigs (p<0.05) at the time of challenge and were protected against CSFV infection, whereas the control pigs died within 14 days post-infection (dpi). In the second experiment, pigs were vaccinated once with either CSFV vaccine or CSFV vaccine combined with Aujeszky's disease (AD) vaccine and challenged at 140 dpv. All vaccinated pigs developed both CSFV-specific, cellular and antibody responses and were protected against CSFV infection. However, differences in cellular, but not antibody, responses were observed in the two vaccinated groups. The group vaccinated with CSFV vaccine developed a significantly higher number of CSFV-specific, IFN-gamma secreting cells (p<0.05), exhibited a shorter fever period and less pathological changes, when compared with the group vaccinated with the combined vaccine. The kinetics of IFN-gamma production, following challenge in the two vaccinated groups, were also different. Taken together, our results indicated that CSFV-specific, IFN-gamma production could be detected early after antigen exposure and correlated with protection against CSFV challenge. Our findings highlight the role of cellular immune responses in porcine anti-viral immunity.
Animals↗
Efficacy and stability of a subunit vaccine based on glycoprotein E2 of classical swine fever virus.
The purpose of this study was to determine the efficacy and stability of an E2 subunit vaccine against classical swine fever virus (CSFV). The vaccine, which contains E2 produced in insect cells by a baculovirus expression vector is a potential marker vaccine, as it allows discrimination between infected and vaccinated pigs. Several vaccination-challenge experiments were performed to determine the dose that protects 95% of the vaccinated pigs (PD95), and to determine the stability and efficacy of the vaccine several months after production. A single vaccination with a vaccine dose of 32 microg E2 - the estimated PD95 - in a water-oil-water adjuvant prevented clinical signs and mortality due to a CSFV challenge-inoculation three weeks after vaccination. Moreover, virus transmission to susceptible sentinel pigs was prevented in nearly all groups of pigs vaccinated with this dose. The vaccine was stable for at least 18 months, and retained its full potency. These findings indicate that the E2 marker vaccine merits further evaluation for suitability for use in a control program during an outbreak of CSF.
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Comparative detection of classical swine fever virus in striated muscle from experimentally infected pigs by reverse transcription polymerase chain reaction, cell culture isolation and immunohistochemistry.
Classical swine fever (CSF) is a highly contagious viral disease, which can be transmitted by CSFV-contaminated swill. In 1993, four CSF outbreaks in Switzerland were caused presumably by feeding pigs with improperly heated swill. The aim of the investigations was to find a suitable method for CSFV detection in striated muscle samples of infected pigs in order to allow routine testing of meat for virus contamination. The sensitivity of virus detection in striated muscle was compared with the detection in target organs. Using reverse transcription polymerase chain reaction (RT-PCR), cell culture isolation and immunohistochemistry on samples from 14 experimentally infected pigs, CSFV was detected in target organs of ten, and in striated muscle of six pigs, respectively. Overall, only 58% of muscle samples from CSFV-positive animals were positive by RT-PCR and 40% by virus isolation in cell culture, whereas the virus was detected in target organs of these pigs. Virus detection from striated muscle was primarily successful in severely diseased animals infected with highly virulent CSFV strains. It is concluded that striated muscle is not suitable for sensitive CSFV detection, and additional organs have to be examined for reliable diagnosis.
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