Phenotypic analysis of peripheral leukocytes in piglets infected with classical swine fever virus.

The phenotypic changes in circulating leukocytes in swine fever influenced by classical swine fever virus (CSFV) infection with different strain virulence was studied in piglets. The phenotypic differences were measured by monoclonal antibodies specific for porcine differentiation antigens. The pattern of phenotypic change varied with the virulence of CSFV. Infection with virulent, but not the attenuated strain of CSFV resulted in the dramatic early loss of CD8-bearing T lymphocytes from the circulation. A similar trend was also seen in the gammadelta T-cell compartment following infection with the highly virulent strain, Washington. The loss of circulating B-lymphocytes was consistent with the failure to generate neutralising antibody. These observations contrasted the finding that the number of leukocytes expressing the CD4 surface antigen increased in piglets infected with CSFV. These data provide preliminary information on the potential range of leukocyte changes produced in piglets following infection with CSFV.     

Virulence of recent and former classical swine fever virus isolates evaluated by their clinical and pathological signs

The clinical diagnosis of classical swine fever (CSF) still caused problems to the veterinarians during the last decade. The primary CSF outbreak was often detected too late and, meanwhile, the virus had spread. Consequently, the recent classical swine fever virus isolates (CSFV) were suspected to be of low virulence. The purpose of the study was to quantify the virulence of four recent CSFV by evaluating the clinical and pathological signs caused by different CSFV. Pigs of the same breed and age group were inoculated intranasally with CSFV from recent epidemics in European Union (EU) member states. The CSFV used are registered in the data base of the EU Reference Laboratory for CSF and belong to different genotypes: 2.1, 2.2 and 2.3 respectively. Clinical signs of CSF were evaluated by using a score system suggested previously (Mittelholzer et al., 2000: Vet. Microbiol. 74, 293). For the evaluation of pathological lesions, a new pathological score was introduced. The four CSFV tested here were classified as moderately virulent in general, although one CSFV may cause different clinical courses, ranging from highly virulent to avirulent. This indicates the importance of additional factors in the host animal for virulence. Differences in the clinical and pathological signs between these four recent CSFV were rather minor, emphasizing that the genetic typing of CSFV is absolutely essential. Differences towards former CSFV (e.g. reference virus strain Alfort 187) were more pronounced, especially regarding the onset and duration of the disease, the occurrence of skin haemorrhages and pathological lesions of kidney, subcutis and serosae. It is concluded that clinical diagnosis of CSF is rather difficult in pigs up to 14 days post-CSFV infection using these four CSFV, emphasizing the need for careful differential diagnosis and the laboratory investigation for CSF at an early stage.     

Generation and efficacy evaluation of a recombinant adenovirus expressing the E2 protein of classical swine fever virus

Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), which causes significant economic losses to the pig industry worldwide. The E2 glycoprotein of CSFV is the main target for neutralizing antibodies. This study was aimed to develop a recombinant human adenovirus type 5 expressing the CSFV E2 gene (rAdV-E2) and evaluate its efficacy in rabbits and pigs. The results showed that the rabbits and the pigs immunized with the rAdV-E2 developed high-level CSFV-specific neutralizing antibodies. The rAdV-E2-immunized rabbits were protected from fever induced by infection with C-strain, which is pathogenic to the rabbit, and the rAdV-E2-immunized pigs were protected from lethal challenge with highly virulent Shimen strain. This indicates that the recombinant adenovirus can be an attractive candidate vaccine for preventing CSF.     

Classical swine fever virus: clinical, virological, serological and hematological findings after infection of domestic pigs and wild boars with the field isolate "Spante" originating from wild boar

A classical swine fever virus (CSFV) field isolate originating from wild boar was investigated on its virulence in domestic pigs and wild boar. Three weaner pigs and two wild boars (yearlings) were intranasally inoculated with the isolate "Spante" and tested for clinical, virological, hematological and serological findings until day 31 after infection (p. i.). One day p. i. the piglets were put in contact to three sentinel pigs. During a period of 31 d neither the domestic pigs nor the wild boars showed clinical signs specific for CSF. Two infected weaner pigs became transiently viraemic, transmitted CSFV in nasal secretions, showed a slight leukopenia and reacted serologically positive. The contact infection resulted in a viraemia in two sentinel piglets on day 30. Only one contact animal developed antibodies. None of the wild boars became viraemic, excreted CSFV in nasal secretions or developed antibodies. The CSFV isolate "Spante" represents a low virulent virus. Referring to a significant higher percentage of virologically positive tissue samples after nested PCR compared with the virus isolation, persistence of CSFV is discussed.     

Limited BVDV transmission and full protection against CSFV transmission in pigs experimentally infected with BVDV type 1b

Bovine viral diarrhea virus (BVDV) in pigs may interfere with the detection and epidemiology of classical swine fever virus (CSFV). To investigate the importance of BVDV infections in pigs, first we studied the transmission dynamics of a recent BVDV field isolate. Subsequently, the protection of BVD antibodies against transmission and clinical disease of CSF virus was studied. Only limited transmission of BVDV occurred (R = 0.20), while no CSFV transmission occurred in pigs with BVDV antibodies. We concluded that BVDV transmission among pigs is possible, but seems to be limited and thus the virus should disappear from a population if no new introductions occur. Furthermore, the presence of BVD antibodies may completely prevent the transmission of CSFV and therefore could protect pigs against classical swine fever. It was also noticed that double infections with BVDV and CSFV were incorrectly diagnosed using the neutralization peroxidase linked assay (NPLA), which is the golden standard for antibody detection. This might hamper the diagnosis of CSF in herds with a high BVD prevalence.     

Efficacy of a live attenuated vaccine in classical swine fever virus postnatally persistently infected pigs

Classical swine fever (CSF) causes major losses in pig farming, with various degrees of disease severity. Efficient live attenuated vaccines against classical swine fever virus (CSFV) are used routinely in endemic countries. However, despite intensive vaccination programs in these areas for more than 20 years, CSF has not been eradicated. Molecular epidemiology studies in these regions suggests that the virus circulating in the field has evolved under the positive selection pressure exerted by the immune response to the vaccine, leading to new attenuated viral variants. Recent work by our group demonstrated that a high proportion of persistently infected piglets can be generated by early postnatal infection with low and moderately virulent CSFV strains. Here, we studied the immune response to a hog cholera lapinised virus vaccine (HCLV), C-strain, in six-week-old persistently infected pigs following post-natal infection. CSFV-negative pigs were vaccinated as controls. The humoral and interferon gamma responses as well as the CSFV RNA loads were monitored for 21 days post-vaccination. No vaccine viral RNA was detected in the serum samples and tonsils from CSFV postnatally persistently infected pigs for 21 days post-vaccination. Furthermore, no E2-specific antibody response or neutralising antibody titres were shown in CSFV persistently infected vaccinated animals. Likewise, no of IFN-gamma producing cell response against CSFV or PHA was observed. To our knowledge, this is the first report demonstrating the absence of a response to vaccination in CSFV persistently infected pigs.     

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.     

Replication of classical swine fever virus strains and isolates in different porcine cell lines

Classical swine fever virus (CSFV) is an economically important pathogen of domestic pigs and wild boar. Due to the highly variable clinical picture of CSF, laboratory methods are essential for an unambiguous diagnosis. Virus isolation using cell culture is still considered the gold standard. It is based on the incubation of permissive cells with organ or leukocyte preparations followed by antigen detection. In the "EU Diagnostic Manual for CSF Diagnosis", the permanent cell line PK(15) (porcine kidney) is recommended. In the European Reference Laboratory (EURL) a clone of this cell line, PK(15)A, and the STE (swine testicular epitheloid) cell line are in use for propagation of CSFV. The aim of this work was to assess the relative ability of eleven permanent cell lines derived from various organs of wild boar and domestic pig, respectively, to support the replication of different strains and isolates in comparison to these cell lines. An avirulent and a highly virulent laboratory CSFV strain, and several recent field isolates from domestic pigs and wild boars were used. Titers were determined after one, two and three virus passages, and after 48 and 120 h of incubation. Of the eleven cell lines analyzed, two were found that replicated all the tested CSFV strains and field isolates. Those may be useful for improving diagnosis of CSFV and for preparing low-passaged virus stocks of new isolates.     

Efficacy of the classical swine fever (CSF) marker vaccine Porcilis Pesti in pregnant sows

The efficacy of the classical swine fever (CSF) subunit marker vaccine Porcilis Pesti based on baculovirus expressed envelope glycoprotein E2 of CSF virus (CSFV) was evaluated in pregnant sows. Ten gilts were vaccinated with one dose of marker vaccine, followed by a second dose 4 weeks later. Four gilts remained unvaccinated and received a placebo at the same times. Thirty-three days after the second vaccination all animals were artificially inseminated. Neither local or systemic reactions nor an increase of body temperature were observed after vaccinations. All gilts showed a normal course of pregnancy. Thirty-five days after first vaccination all animals developed E2 specific neutralising antibodies with titres in the range of 5.0 and 7.5 log(2). No antibodies to CSFV-E(rns) were found in ELISA.On day 65 of gestation (126 days after the first immunisation) all sows were infected intranasally using 2ml (10(6.6) TCID(50)/ml) of the low virulent CSFV strain "Glentorf". After challenge in two of the unvaccinated control sows a slight transient increase of body temperature was observed, whereas leukopenia was demonstrated in all control animals. In addition all controls became viraemic. Vaccinations with the CSFV subunit vaccine protected the animals from clinical symptoms of CSF. In two sows a moderate decrease of leukocyte counts was detected on day 5 post infection. In contrast to the unvaccinated control sows in none of the vaccinated animals virus was isolated from the nasal swabs or the blood.Approximately 40 days after challenge all sows were killed and necropsy was done. The sows and their offspring were examined for the presence of CSFV in blood, bone marrow and different organs. No virus was found in any of the sows. In contrast, in all litters of the control sows CSFV was found in the blood as well as in the organ samples. Nine out of 10 litters of the vaccinated sows were protected from CSFV infection. Blood samples, lymphatic organs and bone marrow of these animals were all virologically negative. When sera were tested for CSFV-antibodies all sows had developed E(rns)-specific antibodies but no CSFV-specific antibodies were found in any of the progeny.It was concluded that vaccination with CSF subunit marker vaccine Porcilis((R)) Pesti protected 90% of the litters from viral infection when sows were challenged mid-gestation using the CSFV-strain "Glentorf".     

Serum antibodies directed against classical swine fever virus and other pestiviruses in wild boar (Sus scrofa) in the Republic of Croatia

The presence of serum antibodies directed against classical swine fever (CSF) virus and other pestiviruses among the wild boar (Sus scrofa) population in Croatia was investigated. During 2003, serum samples from 214 wild boars were collected in 10 hunting areas in the continental part of the country.The sera were examined by enzyme immunoassay (ELISA) and in the virus neutralization test (VNT). Out of 214 sera tested 111 (51.87 %) were positive by ELISA and regarding neutralising antibodies, against CSFV 75 (35.05 %) samples were positive. In the VNT with the C-strain (conventional live vaccine strain China) and the strain Uelzen were used. Samples were also tested for neutralizing antibodies against border disease virus (BDV) using the strain 137/4 and against bovine viral diarrhoea virus (BVDV) using the NADL strain. Neutralizing antibodies against the C-strain were detected in 36 sera (16.82 %), against strain Uelzen in 17 sera (7.94 %) and in 22 sera (10.28 %) against both strains. In five sera (2.33 %) neutralizing antibodies against BVDV and BDV were found.     

Duration of the protection of an E2 subunit marker vaccine against classical swine fever after a single vaccination

The period during which pigs are protected after vaccination is important for the successful usage of a marker vaccine against classical swine fever virus (CSFV) in an eradication programme. In four animal experiments with different vaccination-challenge intervals we determined the duration of protection of an E2 subunit marker vaccine in pigs after a single vaccination. Unvaccinated pigs were included in each group to detect transmission of the challenge virus.Three groups of six pigs were vaccinated once and subsequently inoculated with the virulent CSFV strain Brescia after a vaccination-challenge interval of 3, 51/2, 6 or 13 months. All vaccinated pigs, 16 out of 18, with neutralising antibodies against CSFV at the moment of challenge, 3, 51/2, 6 or 13 months later, survived, whereas unvaccinated control pigs died from acute CSF or were killed being moribund. A proportion of the vaccinated pigs did however develop fever or cytopenia after challenge and two vaccinated pigs were viremic after challenge. Virus transmission of vaccinated and challenged pigs to unvaccinated sentinel pigs did not occur in groups of pigs which were challenged 3 or 6 months after a single vaccination. Two out of eight vaccinated pigs that were found negative for CSFV neutralising antibody at 13 months after vaccination died after subsequent challenge.The findings in this study demonstrate that pigs can be protected against a lethal challenge of CSFV for up to 13 months after a single vaccination with an E2 subunit marker vaccine.     

Classical swine fever virus in plasma and peripheral blood mononuclear cells of acutely infected swine

The distribution of classical swine fever virus (CSFV) in plasma, monocytes, T and B lymphocytes in peripheral blood was monitored during experimentally induced acute classical swine fever infection in piglets. Six piglets were infected with 10(3.8) TCID50 of virus and blood samples taken up to 18 days post-inoculation (p.i.). Infectious virus was detected in monocytes, T and B lymphocytes to similar titres in five of the six infected piglets. Infectious virus was detected earlier in plasma than in any of the mononuclear cell subpopulations. No significant difference was observed in the period of time in which virus could be isolated from the three cell subpopulations. While a progressive lymphopenia developed, a marked B cell depletion was observed. However, B cells were apparently replaced by non-IgM-bearing mononuclear cells, as the proportion 'total lymphocyte/total leucocytes' remained unaltered throughout the experiment. Virus titres in plasma and peripheral blood mononuclear cells showed a tendency to increase as the disease progressed to its outcome.     

Development of a classical swine fever subunit marker vaccine and companion diagnostic test

The development of a classical swine fever (CSF) subunit marker vaccine, based on viral envelope glycoprotein E2, and a companion diagnostic test, based on a second viral envelope glycoprotein E(RNS), will be described. Important properties of the vaccine, such as onset and duration of immunity, and prevention of horizontal and vertical transmission of virus were evaluated. A single dose of the vaccine protected pigs against clinical signs of CSF, following intranasal challenge with 100LD(50) of virulent classical swine fever virus (CSFV) at 2 weeks after vaccination. However, challenge virus transmission to unvaccinated sentinels was not always completely inhibited at this time point. From 3 weeks up to 6 months after vaccination, pigs were protected against clinical signs of CSF, and no longer transmitted challenge virus to unvaccinated sentinels. In contrast, unvaccinated control pigs died within 2 weeks after challenge. We also evaluated transmission of challenge virus in a setup enabling determination of the reproduction ratio (R value) of the virus. In such an experiment, transmission of challenge virus is determined in a fully vaccinated population at different time points after vaccination. Pigs challenged at 1 week after immunization died of CSF, whereas the vaccinated sentinels became infected, seroconverted for E(RNS) antibodies, but survived. At 2 weeks after vaccination, the challenged pigs seroconverted for E(RNS) antibodies, but none of the vaccinated sentinels did. Thus, at 1 week after vaccination, R1, and at 2 weeks, R=0, implying no control or control of an outbreak, respectively. Vertical transmission of CSFV to the immune-incompetent fetus may lead to the birth of highly viraemic, persistently infected piglets which are one of the major sources of virus spread. Protection against transplacental transmission of CSFV in vaccinated sows was, therefore, tested in once and twice vaccinated sows. Only one out of nine once-vaccinated sows transmitted challenge virus to the fetus, whereas none of the nine twice-vaccinated sows did. Finally, our data show that the E(RNS) test detects CSFV-specific antibodies in vaccinated or unvaccinated pigs as early as 14 days after infection with a virulent CSF strain. This indicates that the E2 vaccine and companion test fully comply with the marker vaccine concept. This concept implies the possibility of detecting infected animals within a vaccinated population.     

分子生物学技术在猪瘟诊断中的应用

猪瘟是由猪瘟病毒引起的一种急性、发热性、接触性传染病,可引起各种年龄猪发病.随着对猪瘟病毒研究的深入,猪瘟在一定程度上得到有效的控制.但是近年来,世界各国流行的猪瘟在流行病学、临床症状和病理变化等方面出现了一些新的变化,猪瘟的防控出现了许多新的情况.早已宣布消灭了猪瘟的一些欧洲国家又报道有猪瘟发生.我国猪瘟的发病率亦呈上升趋势,严重威胁着我国养猪业的发展,给养猪业造成极大的经济损失.论文综述了猪瘟分子生物学诊断技术的研究进展,为猪瘟的及时诊断提供参考.     

Yeast-expressed classical swine fever virus glycoprotein E2 induces a protective immune response

Classical swine fever (CSF) is an economically important swine disease worldwide. The glycoprotein E2 of classical swine fever virus (CSFV) is a viral antigen that can induce a protective immune response against CSF. A recombinant E2 protein was constructed using the yeast Pichia pastoris expression system and evaluated for its vaccine efficacy. The yeast-expressed E2 (yE2) was shown to have N-linked glycosylation and to form homodimer molecules. Four 6-week-old specified-pathogen-free (SPF) piglets were intramuscularly immunized with yE2 twice at 3-week intervals. All yE2-vaccinated pigs could mount an anamnestic response after booster vaccination with neutralizing antibody titers ranging from 1:96 to 1:768. Neutralizing antibody titers at 10 weeks post booster vaccination ranged from 1:16 to 1:64. At this time, the pigs were subjected to challenge infection with a dose of 1 × 10⁵ TCID₅₀ (50% tissue culture infective dose) virulent CSFV strain. At 1 week post challenge infection, all of the yE2-immunized pigs were alive and without symptoms or signs of CSF. Neutralizing antibody titers at this time ranged from 1:4,800 to 1:12,800 and even to 1:51,200 one week later. In contrast, the control pigs continuously exhibited signs of CSF and had to be euthanized because of severe clinical symptoms at 6 days post challenge infection. All of the yE2-vaccinated pigs were E^rns antibody negative and had seroconverted against E^rns by post challenge day 11, suggesting that yE2 is a potential DIVA (differentiating infected from vaccinated animals) vaccine. The yeast-expressed E2 protein retains correct immunogenicity and is able to induce a protective immune response against CSFV infection.     

Time-dependent infection probability of classical swine fever via excretions and secretions

Several routes contribute to the spread of classical swine fever (CSF) during outbreaks of this disease. However, for many infected herds in recent epidemics, no route of virus introduction could be indentified. To obtain more insight into the relative importance of secretions and excretions in transmission of CSF virus, a model was developed. This model quantified the daily transmission probabilities from one infectious pig to one susceptible pig, using quantitative data on: (a) virus excretion by infected pigs, (b) survival of virus in the environment and (c) virus dose needed to infect susceptible pigs. Furthermore, the model predicted the relative contribution of secretions and excretions to this daily probability of infection of a susceptible pig. Three virus strains that differed in virulence were evaluated with the model: the highly virulent strain Brescia, the moderately virulent strain Paderborn and the low virulent strain Zoelen. Results suggest that it is highly probable that susceptible pigs in contact with Brescia or Paderborn infected pigs will be infected. For a pig in contact with a Zoelen infected pig, infection is less likely. When contact with blood is excluded, the predicted overall probability of infection was only 0.08 over the entire infectious period. The three strains differed in the relative contribution of secretions and excretions to transmission, although blood had a high probability of causing infection of a susceptible pig when in contact with a pig infected with any strain. This supports the statement that during outbreaks, control measures should ideally be based on the characteristics of the specific virus strain involved, which implies the development of strain-specific measures.