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Induction of apoptosis in bone marrow neutrophil-lineage cells by classical swine fever virus.

The pathogenesis of bone marrow atrophy during classical swine fever (CSF) was investigated in vitro by using CSF virus (CSFV) infection of bone marrow haematopoietic cells (BMHC). The monocytic lineage had the highest susceptibility to CSFV infection, whereas the more mature SWC8(+) granulocytic cells were not directly susceptible to infection. However, myelomonocytic precursors were targets for CSFV infection and continued to differentiate into SWC8(+) granulocytic cells, which remained infected. This explains the occurrence of infected peripheral blood granulocytes during CSF. The infection of BMHC resulted in increased apoptosis and necrosis, mainly within the granulocytic lineage. Caspases 3 and 9 were particularly activated, relating to the mitochondrial pathway of apoptosis. Interestingly, the majority of infected cells were non-apoptotic, the apoptotic cells being primarily non-infected. This indicated an indirect mechanism for induction of apoptosis, but no role could be identified for bone marrow stroma cells such as macrophages or fibroblastoid cells. Furthermore, soluble factors including cytokines and reactive oxygen species were not primarily responsible. In contrast, contact between infected and non-infected BMHC was critical for increasing apoptosis in the latter. Taken together, these results in vitro relate to and help to explain further the apoptosis of BMHC that occurs in vivo during CSF. This experimental system will also be particularly useful for the study of CSFV gene products involved in leukocyte apoptosis.

Animals↗

An experimental infection (II) to investigate the importance of indirect classical swine fever virus transmission by excretions and secretions of infected weaner pigs.

An experiment was set up to investigate the role of excretions and secretions in the indirect transmission of classical swine fever virus (CSFV). In five small pens, 10 weaner pigs (two pigs per pen) were housed and inoculated with CSFV. Experimental infection was successful in all pigs. The infected pigs were kept in the pens for a period of 15 days after which the pens were depopulated and pigs were killed. At the moment of depopulation, all inoculated pigs were visibly clinically diseased and had high fever. Ten hours later the same pens were repopulated with five pairs of susceptible pigs. From inoculation onwards and especially between depopulation and restocking, the pens were neither cleaned nor disinfected. Four days post-repopulation, three of the susceptible pigs were detected positive on virus isolation. A fourth pig was detected positive 2 days later. Later on, the remaining pigs also became infected, most probably due to contact and between pen infections. It can be concluded that transmission of the virus via excretions and secretions succeeded in four of 10 pigs. This result indicates that transmission of CSFV via excretions and secretions can be of importance in a late, clinical stage of disease.

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Localization of classical swine fever virus in male gonads during subclinical infection.

In an experiment using ten boars, the distribution of classical swine fever virus (CSFV) was determined in the male reproductive tract by in situ hybridization over a period of 120 days after intranasal inoculation. CSFV was detected in the testicular tissue of infected boars. Viral nucleic acid was localized to spermatogonia, spermatocytes and spermatids but was not detected in the epithelia of the prostate, epididymis or bulbourethral gland. Sections from control, CSFV-negative, pigs showed no hybridization signals for CSFV. The demonstration that CSFV infects the spermatogonia (and their progeny) suggests that this may serve as a primary reservoir for the venereal spread of CSFV.

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[The situation of classical swine fever in wild boars in the European community and selected aspects of disease transmission].

The situation of classical swine fever (CSF) in Europe is described on the basis of the literature. In the European Community, CSF is present among wild boars in Germany (Federal states Mecklenburg-Western Pomerania, Brandenburg and lower Saxony), in France (Northern Vosges) and in Italy (Regio Emìlìa Romagna in 1997 and Sardinia--enzootically infected). Infected wild boars are important as a source of infection for domestic pigs in Germany. Selected aspects of the transmission of CSF virus from wild boars to domestic pigs and within the wild boar population are discussed. Measures for the eradication of CSF in wild boars are described briefly.

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Classical swine fever: morphological and morphometrical study of pulmonary intravascular macrophages.

To gain further insight into the pathogenesis of classical swine fever (CSF), the changes induced by hog cholera (HC) virus in pulmonary intravascular macrophages (PIMs) were examined. Twelve pigs were inoculated by the intramuscular route with a virulent strain of HC virus (Quillota strain) and killed in groups of three at 4, 7, 10 and 14 days post-inoculation. Immunohistochemical and ultrastructural examination revealed HC virus infection in endothelial cells, PIMs, and interstitial and alveolar macrophages. In addition to viral replication, a predominant feature was the secretory activation of PIMs, characterized by expanded rough endoplasmic reticulum and hyperplastic Golgi complexes. The results obtained suggest that macrophage activation and the subsequent release of pro-inflammatory mediators play an important role in the pathogenesis of CSF.

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Analysis of classical swine fever virus replication kinetics allows differentiation of highly virulent from avirulent strains.

To study the replication of classical swine fever virus (CSFV) in cell culture, kinetics of viral plus-strand RNA synthesis, of viral structural and non-structural protein expression as well as of secreted and cell-associated infectious virus were determined. Highly virulent, moderately virulent and avirulent strains that were tested in standardized animal experiments to confirm their virulence were used to search for in vitro parameters allowing the differentiation of strains according to their virulence. No significant qualitative or quantitative differences were found between the strains studied when either RNA replication or protein synthesis were investigated. However, the ratio of cell-associated virus versus secreted virus proved to be considerably lower for the highly virulent strains when compared to avirulent or moderately virulent strains. These data suggest that highly virulent strains of CSFV can be distinguished in cell culture from strains with reduced virulence.

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[Differential diagnosis of classical swine fever and border disease: seroepidemiological investigation of a pestivirus infection on a mixed sheep and swine farm].

During recent years neutralizing antibodies against Border Disease Virus (BDV) were found repeatedly in German pig herds. Consequently there was a demand for a differential diagnostic system. A permanent sheep cell line and BDV reference strain Moredun were chosen and were applied in a could be used case study. A pestivirus could be isolated from piglets on a mixed farm and was characterised as 'non-Classical Swine Fever' (CSF) by using monoclonal antibodies. Due to a CSF suspicion the pig herd was destroyed immediately. Serum samples of sheep from the same farm were used for further characterisation of the new virus isolate. A neutralization test of the sheep sera was performed against different pestiviruses and the new isolate. Neutralizing antibody titres against the new virus pig isolate were significantly higher than against all other pestiviruses. BDV strain Moredun recognised the antibodies clearly, whereas CSF viral strain Alfort 187 and several isolates of bovine viral diarrhoea virus (BVDV) strains scored the lowest cross reaction.

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Prophylactic application of Propionibacterium avidum KP-40 in swine with acute experimental infections. I. Viral infections--Aujeszky's disease and classical swine fever.

The usefulness of the prophylactic application of Propionibacterium avidum KP-40 (PA), a potent stimulator of the macrophage-monocyte system and inducer of endogenous interferons, was demonstrated in swine infected experimentally with Aujeszky's disease or classical swine fever viruses. Some of the infected animals were preimmunized with respective vaccines containing live, attenuated viruses. In vaccinated and non-vaccinated swine infected with Aujeszky's disease virus, pretreatment with PA lowered the morbidity rate, shortened the period of fever and fastened the recovery. Infection with classical swine fever virus resulted in 100% mortality of PA-pretreated non-vaccinated swine, but the length survival of the animals was significantly longer (p < 0.05).

Adjuvants, Immunologic↗

Classical swine fever (CSF): a historical review of research and vaccine production on the Isle of Riems.

A review on classical swine fever (CSF) research and vaccine production is given about four historical periods (1924-1948, 1949-1969, 1970-1991, since 1992). Similar as to research on foot and mouth disease, applied topics as diagnosis, pathogenesis, epidemiology and control represented the CSF research over many years. The development of vaccines and application procedures, e.g. oral and aerogenic immunisation and combined vaccines for large pig farms were the prominent investigations between 1950 and the middle of 1980s. After being reduced in the first years after affiliation to the Federal Research Centre for Virus Diseases of Animals, CSF is one of the main topics of the research on the Isle of Riems, not at least because nowadays the German National Reference Laboratory for CSF was established on the Island.

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Classical swine fever in wild boar in the Lombardy region of Italy from 1997 to 2002.

In 1997, outbreaks of classical swine fever occurred in wild boar and domestic pigs in the Italian province of Varese (Lombardy region). Statutory animal health measures were imposed, and a control plan aimed at preventing the virus from spreading outside the infected hunting zone of 108 km2 was implemented. However, in 1998, virologically positive wild boar were found outside this zone, including within Swiss territory. The infected zone was then enlarged to include the three other hunting areas of the northern province of Varese and the neighbouring Swiss territory. Italian and Swiss veterinary services initiated a joint policy to control the disease by hunting young wild boar and conserving older, immune animals. The percentage of virologically positive animals gradually decreased, the last being found in July 2000. The seroprevalence initially increased, but in 2000 and 2001 it decreased from 42.2 per cent to 8.8 per cent, indicating that the wild boar population had had no further contact with the virus.

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Quantitative assessment of clinical signs for the detection of classical swine fever outbreaks during an epidemic.

The performance of clinical signs as a diagnostic test for the detection of classical swine fever (CSF) outbreaks during the 1997-1998 CSF epidemic in The Netherlands was evaluated by constructing and analysing a receiver operating characteristic (ROC) curve. This curve assesses the discriminating ability of a diagnostic test over a range of test signals. The cut-off values for a defined diagnostic test to detect CSF outbreaks were set by different combinations of clinical signs observed. The area under the ROC curve, which is a quantitative measure of test performance, was significantly (P<0.001) larger than the area under the random ROC curve. This indicates that clinical signs have a significantly higher performance as a diagnostic test for the detection of CSF than for flipping a coin. However, the gain in diagnostic performance compared to a random process is not as much as we would wish it to be. The optimal efficient diagnostic test combined a sensitivity of 72.7% with a specificity of 52.7%, with a combination of the following clinical signs: unsteady gait/ataxia, not eating, not reacting to antibiotic treatment, conjunctivitis, hard faecal pellets.

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Within-farm spread of classical swine fever virus--a blueprint for a stochastic simulation model.

A stochastic simulation model to investigate the transmission of classical swine fever (CSF) virus within an infected farm is described. The model is structured according to the processes that occur within and between management groups (pig units or houses). It uses the individual pig as the unit of interest and estimates the number of animals in the states 'susceptible', 'infected', 'infectious', and 'removed' for each day of the disease incident. Probabilities are assigned to the transitions between states. The probability of a pig becoming infected is made dependent on the probability of contact between a susceptible and an infectious pig as well as the probability of transmission. The more pigs become infected in one unit, the more likely is subsequent spread to another management group on the farm. Ultimately, the probability that a shipment of pigs from the farm will include at least one infected pig can be estimated in order to identify high-risk movements during a CSF epidemic. The model results were compared with experimental data on CSF transmission within one pig unit (management group). It could be shown that the model was capable of reproducing the experimentally observed infection and mortality rates. To improve the input parameters and for further model validation, more experimental data and field data from CSF outbreaks are needed.

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Classical swine fever (CSF) marker vaccine. Trial II. Challenge study in pregnant sows.

The efficacy of two marker vaccines against classical swine fever (CSF) was tested in a large scale laboratory trial in several National Swine Fever Laboratories (NSFL) of the EU member states. The vaccines were: BAYOVAC CSF Marker (Vaccine A) from Bayer, Leverkusen, Germany and PORCILIS PESTI (Vaccine B) from Intervet, Boxmeer, The Netherlands. At the NSFL of Belgium, The Netherlands and Germany experiments were carried out to examine the ability of the vaccines to prevent transplacental transmission of CSF virus. In Belgium and The Netherlands pregnant sows were vaccinated once and challenged with virulent CSF virus 14 days later, which was around day 60 of gestation. At the NSFL in Germany sows were vaccinated twice, on days 25 and 46 of pregnancy and were challenged fourteen days after booster vaccination (day 60 of gestation). Apart from minor inflammatory reactions in some sows, no reactions post vaccination were noticed in either vaccine group. Sows vaccinated with Vaccine A were better protected against clinical CSF than sows vaccinated with Vaccine B. The antibody response after vaccination with Vaccine A was more pronounced than after vaccination with Vaccine B. After single vaccination six out of eight sows vaccinated with Vaccine A and all eight sows vaccinated with Vaccine B had viraemic piglets. After double vaccination one out of four litters from sows vaccinated with Vaccine A and four out of five litters from sows vaccinated with Vaccine B were found to be viraemic. However, both vaccines reduced the transmission probability significantly (Vaccine A: P=0.004, Vaccine B: P=0.024) after booster vaccination. However, Vaccine A appeared in this regard more potent as the estimated probability of fetal infections was lower. Nevertheless the risk of virus spreading after vaccination via transplacental transmission is still present and has to be addressed from an epidemiological point of view.

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Epidemiological characteristics of an outbreak of classical swine fever in an area of high pig density.

The objective of this study was to analyse an outbreak of classical swine fever under a policy of non-vaccination, intensive surveillance and eradication in an area of high pig density. The virus was found in 52 herds, where some 90,000 pigs were slaughtered. The clinical signs were vague and the reports of suspect herds generally coincided with increased mortality. The interval between the first occurrence of clinical signs and the report of a suspect herd was shorter when the disease was first diagnosed in fattening pigs than when it was diagnosed in sows, boars or suckling piglets. Among fattening pigs, mortality and morbidity appeared to increase with age. The proportion of clinically ill animals was positively correlated with the proportion of serologically positive animals in a pig house during the phase when the disease was spreading. Fifty-eight per cent of pig houses containing only clinically healthy but some virologically positive pigs were serologically negative. Antigen detection was therefore critical for early disease detection. Serology was nevertheless useful to ascertain that swine fever was not endemic in the area. The secondary cases were concentrated in the close neighbourhood of the herd initially infected.

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Prevention of transplacental transmission of moderate-virulent classical swine fever virus after single or double vaccination with an E2 subunit vaccine.

The use of a vaccine against classical swine fever virus (CSFV) during an outbreak of CSF should lead to a reduction in the horizontal or vertical transmission of CSFV. The reduction of vertical, i.e. transplacental, transmission of a moderate-virulent strain of CSFV from the sow to its offspring was studied in sows vaccinated once or twice with a CSFV E2 subunit vaccine. Two groups of nine sows were vaccinated with one PD95 dose of the E2 subunit vaccine, approximately four weeks before insemination. A third group of nine inseminated sows served as controls. One group of nine sows were vaccinated again at two weeks after insemination. At ten weeks after the primary vaccination, approximately six weeks after insemination, all 27 sows were challenged intranasally with 10(5) TCID50 of a moderate-virulent strain of CSFV, the Van Zoelen strain. The sows were euthanized at five weeks after challenge, and samples from the sows and fetuses were collected for detection of CSFV. All 27 sows were in gestation at the time of slaughter, CSFV was detected in the fetuses of all unvaccinated sows but it was not detected in any of the samples collected from fetuses of the double-vaccinated sows. Virus was however recovered from the fetuses of one out of nine sows vaccinated once. All the sows, except four double-vaccinated sows, developed CSFV Erns antibodies. Transplacental transmission of CSFV was reduced significantly (p <0.001) in all vaccinated sows. When the results from the experiment were extrapolated to a herd level, it could be concluded that, with 95% certainty, approximately 11% (single vaccination) or 0% (double vaccination), confidence intervals of 0.01-0.44 and 0.0-0.30 respectively, of the pregnant sows would still not be protected against vertical transmission of moderate-virulent CSFV. We conclude that vaccination with the CSFV E2 subunit vaccine can reduce the transmission of moderate-virulent strain of CSFV from the sow to its offspring significantly.

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[Classical swine fever].

The principles of aetiology, pathogenesis, diagnosis and immunology of classical swine fever as well as control measures are described. This review focuses on new diagnostic methods, immune reactions and possibilities for control of CSF in the context of the current eradication plans.

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