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PubMed2003

Pathogenicity and kinetics of virus propagation in swine infected with the cytopathogenic classical swine fever virus containing defective interfering particles.

To analyze the pathogenicity and in vivo kinetics of the cytopathogenic (cp) classical swine fever virus (CSFV) WB82 strain, which is composed of cp defective interfering (DI) particles and noncytopathogenic (noncp) helper virus (WB82/E(+) strain), WB82 and WB82/E(+) strains were administered separately to domestic pigs. After inoculation with either strain, all pigs showed typical symptoms of classical swine fever (CSF), such as leucopenia and high fever. There were few differences in clinical signs and survival times between each group. However, the appearance of some symptoms of CSF had a tendency to be delayed following infection with the WB82 strain, when compared with the WB82/E(+) strain. Virus isolation and detection of subgenomic (sg) and full-length viral (flv) RNA by RT-PCR was carried out using sera, 10% homogenized organs and oral, nasal and rectal swabs. Both noncytopathogenic helper virus and cp DI particles were detected in samples from pigs infected with the WB82 strain, but only noncp phenotype virus was isolated from pigs infected with the WB82/E(+) strain. Interestingly, the cp DI particles appeared six to seven days later than helper virus in sera from pigs infected with the WB82 strain. Although active cp DI particles could not be isolated from swabs, sg RNA as well as flv RNA was detected in swabs from animals infected with the WB82 strain. These results suggest that progeny cp DI particles are replicated from parent DI particles after noncp virus replication, and subsequently discharged from infected animals. Furthermore, propagation of DI particles or replication of sg RNA, following propagation of helper virus, appears to inhibit the appearance of CSF symptoms induced by virulent helper CSFV.

Animals↗

PubMed2005

Diagnostic evaluation of a real-time reverse transcriptase PCR assay for detection of classical swine fever virus.

A fluorogenic-probe hydrolysis (TaqMan)-reverse transcriptase (RT) PCR for classical swine fever virus (CSFV) was evaluated for diagnostic sensitivity and specificity by using clinical samples obtained from the Dominican Republic, where the disease is enzootic. The sensitivity of this test, using nasal swab samples taken from both symptomatic and asymptomatic animals, exceeded the diagnostic sensitivity of virus isolation (100% versus 72.4%, respectively) with little loss of specificity (98.9% versus 100%, respectively). At the herd level, three of four infected farms were identified by virus isolation, while the CSFV real-time RT-PCR assay identified all four infected premises. This simple and accurate test permits rapid detection of CSFV in affected herds.

Animals↗

PubMed2001

DNA-mediated protection against classical swine fever virus.

Four eukaryotic expression plasmids containing the entire E2 gene sequence of classical swine fever virus (CSFV) were constructed: (a) pcDST, with 5' signal and 3' transmembrane sequences; (b) pcDSW, with 5' signal sequence only; (c) pcDWT, with transmembrane sequences only; and (d) pcDWW, containing the E2 gene alone. All four plasmids were readily transfected into BHK-21 cells, with pcDST and pcDSW resulting in secretion of E2 antigen. The latter two plasmids were also shown to induce a humoral immune response against CSFV in mice when administered intramuscularly, but no immune responses were detected with either pcDWT or pcDWW. The antibody level elicited by pcDSW was higher than that induced by pcDST. When pcDSW was used to immunize rabbits and pigs, both species were shown to be protected from challenge with virulent CSFV (hog cholera lapinized virus for rabbits and Shimen strain for pigs).

Animals↗

PubMed2000

Molecular epidemiology of a large classical swine fever epidemic in the European Union in 1997-1998.

A big epidemic of classical swine fever (CSF) occurred in the European Community in 1997. The first case was reported at the beginning of January 1997 from Germany. The disease presumably spread to the Netherlands, and from there to Italy, Spain and eventually to Belgium. About 30 isolates from these outbreaks were analysed by comparison of the nucleotide sequence data generated from fragments of both the E2 glycoprotein gene (190 nucleotides) and from the 5'-nontranslated region (5'-NTR; 150 nucleotides). By combining epidemiological data with genetic typing, it was found that the outbreaks were related and caused by a virus belonging to the genetic subgroup 2.1. As this type of virus had been reported infrequently in Europe and not at all since 1993, we postulate that it was newly introduced into the European Union (EU).

5' Untranslated Regions↗

PubMed2005

Phylogenetic analysis of classical swine fever virus in Taiwan.

Two envelope glycoprotein (Erns and E2) regions of the classical swine fever virus (CSFV) were amplified by RT-PCR and sequenced directly from 158 specimens collected between 1989 and 2003 in Taiwan. Phylogenetic analysis of the two regions revealed a similar tree topology and the Erns region provided better discrimination than the E2 region. One hundred and fifteen isolates out of the 158 isolates were clustered within subgroup 2.1 (further classified as 2.1a and 2.1b) and 2.2, which were considered to be likely of the introduced strains, whereas the remaining 43 isolates were clustered within subgroup 3.4 and were considered to be of the endemic strains. The subgroup 2.1a viruses were first detected in 1994 and predominated from 1995 onwards. However, subgroup 3.4 viruses were prevalent in the early years, not being isolated after 1996. We have observed a dramatic switch in genotype from subgroup 3.4 to 2.1a. The subgroup 2.1a isolates are closely related to the Paderborn and Lao isolates, whereas 2.1b isolates have a close relationship to the Chinese Guangxi isolates. The phylogenetic tree of 27 CSFV sequences based on the complete envelope glycoprotein gene (Erns-E2) displayed better resolution than that based on the complete open reading frame.

Animals↗

PubMedDate not supplied

Humoral and cell-mediated immune responses of d/d histocompatible pigs against classical swine fever (CSF) virus.

A better understanding of cell-mediated immune responses to classical swine fever virus (CSFV) is essential for the future development of improved vaccines. We analyzed the generation of cell-mediated and humoral immune responses in d/d histocompatible pigs following CSFV infection or vaccination. Viral infection induced high T cell responses with high primary and secondary CTL activity correlated with high IFN-gamma production, whereas vaccination with a live vaccine followed by infection mainly induced neutralizing antibody but low cell-mediated responses. Moreover, high IgG1 response was associated with high IFN-gamma response following infection whereas a weak IFN-gamma response was related to a good IgG2 response but a low IgG1 production. These data could reflect Th1/Th2-like balance of immune responses depending upon immunization protocols, which has not yet been described in the pig. T-cell responses to CSFV were evidenced by CSFV-specific CD25 upregulation on CD4-CD8+, but not on CD4+CD8- cells, which further illustrated the importance of CTL responses after infection. Our results indicated that generation of cell-mediated immune responses was much higher following intranasal/oral CSFV infection than after intramuscular vaccination, which implies that the capacity of new CSFV vaccines to induce higher T-cell responses should be considered.

Animals↗

PubMed1998

A novel approach to the detection of classical swine fever virus by RT-PCR with a fluorogenic probe (TaqMan).

Detection of classical swine fever virus (CSFV) and its discrimination from other pestiviruses can be achieved by virus isolation (VI) in cell cultures, antigen detection, or molecular analysis. To simplify the latter, a 5'-nuclease assay (TaqMan) was developed for the rapid and specific detection of CSFV with the minimum of downstream PCR processing. A pair of 5'-non-coding region, panpestivirus-specific PCR primers were assessed in a one-step reverse transcription-PCR with each of 36 diverse pestiviruses. The PCR products were subsequently reamplified, in conjunction with a CSFV-specific fluorogenic probe, in a nested-PCR with a second set of panpestivirus PCR primers. During nested PCR, when the target of interest was present, the CSFV probe annealed to the amplicon between the forward and reverse primers and was subsequently cleaved via the 5'-3' nucleolytic activity of the DNA polymerase resulting in the release of the fluorescent reporter dye. Each PCR tube was then placed directly into a luminescence spectrometer to monitor for any increase in fluorescence due to cleavage of the probe. This assay detected representatives of all genetic sub-groups of CSFV, but gave negative results for other pestiviruses. A preliminary assessment showed that the method could be used to detect CSFV RNA extracted from infected pig blood with a sensitivity greater than that of VI.

Age Factors↗

PubMed2004

Scenario tree modeling to analyze the probability of classical swine fever virus introduction into member states of the European Union.

The introduction of classical swine fever virus (CSFV) into a country free of disease without vaccination may have huge consequences in terms of both disease spread and economic losses. More quantitative insight into the main factors determining the probability of CSFV introduction (PCSFV) is needed to optimally use resources for the prevention of CSFV introduction. For this purpose a spreadsheet model was constructed that calculates the annual PCSFV into member states of the European Union (EU). The scenario pathway approach was used as most probabilities in the model are very small. Probability distributions were used to take into account inherent variability of input parameters. The model contained pathways of CSFV introduction including the import of pigs and pork products, returning livestock trucks, and contacts with wild boar. All EU member states were included as possible sources of CSFV. Default results for the Netherlands showed a mean overall annual PCSFV of approximately 0.06, indicating that the Netherlands can expect CSFV introduction on average once every 18 years from the pathways and countries included in the model. Almost 65% of this probability could be attributed to the pathway of returning livestock trucks. The most likely sources of CSFV introduction were Germany, Belgium, and the United Kingdom. Although the calculated probabilities were rather low when compared with expert estimates and recent history, the most likely causes of CSFV introduction indicated by the model were considered to be realistic. It was therefore concluded that the model is a useful tool to structure and analyze information for decision making concerning the prevention of CSFV introduction.

Animals↗

PubMed1999

Laboratory decision-making during the classical swine fever epidemic of 1997-1998 in The Netherlands.

The National Reference Laboratory for classical swine fever (CSF) virus in The Netherlands examined more than two million samples for CSF virus or serum antibody during the CSF epizootic of 1997-1998. The immense amount of samples and the prevalence of border disease (BD) virus and bovine viral diarrhoea (BVD) virus infections in Dutch pig herds necessitated the diagnostic efforts of the laboratory to be focused on generating CSF specific test results throughout the eradication campaign. Detection of 82% of the 429 outbreaks was achieved through the combined use of a direct immunofluorescence and peroxidase assay (FAT/IPA) with samples (tonsils) collected from clinically-suspected pigs. This suggests that in the majority of the outbreaks, the pigs had clinical signs that were recognised by the farmer and/or veterinarians, indicating the presence of CSF virus in a pig herd. A positive diagnosis of 74% of all the tissue samples (tonsils) collected at infected pig holdings was established by FAT. More than 140,000 heparinised blood samples were examined by virus isolation, resulting in the detection of 4.5% of the infected herds. CSF virus was isolated in approximately 29% of all the blood samples collected from pigs at infected or suspected farms. Several serological surveys--each done within a different framework--led to the detection of 13.5% of the total number of outbreaks. The detection of CSF virus antibody in serum was carried out by semi-automated blocking ELISA. Approximately 28.5% of the sera which reacted in the ELISA were classified as CSF virus-neutralising antibody positive and 26.5% as positive for other pestiviruses following the virus neutralisation test (VNT). We concluded that two of the CSF laboratory diagnostic methods described were determinative in the eradication campaign: first, the FAT for the screening of diseased pigs; and second, the ELISA and VNT when millions of predominantly healthy pigs needed to be screened for the presence of CSF serum antibody. Decision-making on the basis of results generated by either method can, however, be seriously hindered when samples are examined from pig herds with a high prevalence of non-CSF pestiviruses.

Animals↗

PubMed2000

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".

Animals↗

PubMed2002

Early platelet aggregation as a cause of thrombocytopenia in classical swine fever.

Twenty pigs were inoculated with a virulent classical swine fever virus isolate to determine the mechanism responsible for thrombocytopenia using histopathologic, ultrastructural, and immunohistochemical (detection of viral antigens gp55 and FVIII-rag) techniques. In animals euthanatized at 2, 4, and 6 days postinoculation (dpi), clusters of granular material staining positive for FVIII-rag were observed in splenic cords, the marginal zone, hepatic sinusoids, and the perisinusoidal space. Moreover, numerous macrophages in these areas were swollen and displayed an intensely positive granular and cytoplasmic reaction. Cell alterations indicative of platelet activation and secretory and phagocytic activation of resident macrophages were also observed in these sites at 2 and 4 dpi. These results suggest that the thrombocytopenia observed in pigs is caused in the first instance by massive activation and subsequent phagocytosis of platelets secondary to the release of platelet-activating factors by activated macrophages.

Animals↗

PubMed1996

Molecular cloning and nucleotide sequence determination of three envelope genes of classical swine fever virus Taiwan isolate p97.

A strain of classical swine fever virus (CSFV) has been isolated in Taiwan. The cDNA coding for three envelope glycoproteins E1, E2 and E3 were molecularly cloned from purified viral particles using the reverse transcription-polymerase chain reaction (RT-PCR) method and sequence-specific primers. The resulting PCR products (1113 bp for E1. 699 bp for E2 and 567 bp for E3) were cloned into the SmaI site of pUC19 and then subjected to DNA sequence analysis. Data showed that nucleotide sequence of the three envelope genes shared a 82-83% homology with the corresponding genes of three other strains (Alfort, Brescia and Weybridge). However, the homology of the deduced amino acid sequence was greater than 90% among the four strains. The potential asparagine-linked glycosylation sites for E1 (5 sites), E2 (7 sites) and E3 (2 sites) were conserved. This suggests that the Taiwan p97 strain is distinct from other three strains described. The variations may have implications for future vaccine development. The sequence has been submitted to GenBank. The accession numbers are U43924 and U03290.

Amino Acid Sequence↗

PubMed2004

The effectiveness of classical swine fever surveillance programmes in The Netherlands.

Consequences of classical swine fever (CSF) epidemics depend on the control measures, but also on the number of infected herds at the end of the high-risk period (HRP). Surveillance programmes aim to keep this number as low as possible, so the effectiveness of surveillance programmes can be measured by the number of infected herds at the end of the HRP. In this paper, an evaluation of the effectiveness of the following five Dutch CSF surveillance programmes is presented: (1) routine gross pathology of severely diseased pigs; (2) routine virological tests of tonsils of all pigs, submitted under 1; (3) daily clinical observation by the farmer; (4) periodic clinical inspection by a veterinarian; (5) leucocyte counts in blood samples from diseased animals on a herd where antimicrobial 'group therapy' is started. The evaluation was done by a modelling study, in which virus transmission, disease development, and actions and diagnostic tests in surveillance programmes were simulated. Also, the yearly costs of the programmes were calculated, and direct costs of CSF epidemics were related to the number of infected herds at the end of the HRP. It appeared that the current Dutch surveillance programmes, without the leucocyte counts, keep the number of infected herds at the end of the HRP below 20 with 95% probability. Leaving out the most-expensive programme of periodic inspection (12.5M per year) does not change this result - indicating that (for CSF surveillance) the programme could well be stopped. If the leucocyte programme, which is currently not effective due to the low sample submission rate, optimally were applied, the 95th percentile could be reduced to 10 infected herds. However, whether application is beneficial is unclear, because of uncertainty of the economic benefits due to the many expected false-positive herds each year.

Animal Husbandry↗

PubMed2005

[Recombinant analysis of classical swine fever virus].

To study the possible recombinant relationship among differently derived classical swine fever virus, the coding regions of 21 isolates were analyzed to detect recombination and breakpoints through gene trees comparison and quartet analyses. The results show nucleotide area corresponding to E0, E1 and E2 as a possible recombinant tract between ALD ( D49532) and GPE-(D49533) while NS5A-NS5B of the isolate 39 (AF407339) appears to be derived from a virulent Shimen strain (AF092448) sequence. This suggests that intertypic exchanges of genetic materials during mixed infections under natural or laboratorial conditions can lead classical swine fever virus to adapt to the changes of host environment.

Classical Swine Fever Virus↗

PubMed2004

Situation of classical swine fever and the epidemiologic and ecologic aspects affecting its distribution in the American continent.

Classical swine fever (CSF) is a viral transboundary animal disease that is highly contagious among domestic and wild pigs, such as boars and peccaries. Today, far from being what was classically described historically, the disease is characterized as having a varied clinical picture, and its diagnosis depends on resorting to proper sample collection and prompt dispatch to a laboratory that can employ several techniques to obtain a definitive diagnosis. Laboratory findings should be complemented with a field analysis of the occurrence of disease to have a better understanding of its epidemiology. The disease is still present in various regions and countries of Latin America and the Caribbean, thus hindering production, trade, and the livestock economy in the region. Consequently, it is among the diseases included in List A of the Office International des Epizooties (OIE). Currently, there are epidemiologic and ecologic aspects that characterize its geographical distribution in the region such as: continued trends in the demand for pork and pork products; an increase in swine investment with low production costs which are able to compete advantageously in international markets; the convention of associating CSF in the syndrome of "swine hemorrhagic diseases" owing to the historical description of its acute presentation and not to the new and more frequent subacute presentations or the diseases with which it may be confused (notably, porcine reproductive and respiratory syndrome and porcine dermopathic nephropathy syndrome, among others); dissemination of the virus through asymptomatic hosts such as piglets infected in utero; frequent lack of quality control and registration of vaccines and vaccinations; feeding of swine with contaminated food waste (swill); the common practice of smuggling animals and by-products across borders; the backyard family production system or extensive open field methods of swine rearing with minimal input in care and feeding; poor understanding of the epidemiologic role that boars and peccaries could have in the transmission and maintenance of the disease in the Americas; and new procedures in animal welfare that some countries are adopting for the production, transport, and slaughter of domestic animals. Consequently, many countries (i.e., Canada, USA, Chile, Belize, Costa Rica, Panama, and Mexico, where 13 of 32 States are disease free) have given priority to the control and progressive eradication of CSF. In other parts of the Americas, the disease appears under control, as is the case of the five countries of the Andean Region and the 12 northern States of Brazil. In South America, Chile, Uruguay and 13 States in Brazil are disease free. Argentina has mounted a national campaign and is in the process of eradicating the disease. No recent information on its presence or distribution in Paraguay is available. With no master strategy to harmoniously progress in the control and eradication of the disease, 17 countries of the region, jointly led by the Food and Agriculture Organization of the United Nations, developed the Continental Plan for the Eradication of CSF whose objective is expected to be reached by 2020.

Animals↗

PubMed2001

Classical swine fever (CSF) marker vaccine. Trial I. Challenge studies in weaner pigs.

Two commercial marker vaccines against classical swine fever virus (CSFV) and companion diagnostic tests were examined in 160 conventional pigs. To test the vaccines in a "worst case scenario", group of 10 weaners were vaccinated using a single dose of an E2 (gp55) based vaccine at days -21, -14, -10 or -7, and subsequently challenged at day 0. The challenge virus was CSFV 277, originating from a recent outbreak of classical swine fever (CSF) in Germany. In all groups, only 5 out of 10 pigs were challenged; the remaining 5 pigs served as vaccinated contact controls. Also, three control groups, each consisting of 10 non-vaccinated pigs, were challenged in parallel to the vaccinated animals. CSFV could be isolated from all non-vaccinated pigs. Among these pigs 40% displayed a chronic course of the infection (virus positive for more than 10 days). Pigs vaccinated 21 or 14 days before challenge displayed no clinical signs of CSFV after challenge. However, they were still able to replicate CSFV when challenged, as measured by reisolation of CSFV from leukocytes of the directly challenged pigs. CSFV could be isolated from the leucocytes of 25% of the pigs vaccinated 21 days before challenge and 50% of the pigs vaccinated 14 days before challenge. Chronic infection was not observed, but transmission to one vaccinated contact pig occurred. From all pigs vaccinated 10 or 7 days before challenge, CSFV could be reisolated. We observed a chronic course of infection in 5% of pigs vaccinated 10 days before challenge and in 30% of pigs vaccinated 7 days before challenge. The mortality rate was 20% in the pigs vaccinated 10 days before challenge, and varied between 20 and 80% in pigs vaccinated 7 days prior to challenge. The contact animals had lower mortality (0-20%) than directly challenged pigs, probably mirroring the delayed time point of infection. There was thus some protection against clinical illness by both marker vaccines, but not a solid protection against infection and virus shedding. The efficacy of the vaccine was best if used 3 weeks before challenge and a clear correlation between time interval from vaccination to challenge and the level of virus shedding was observed. Each vaccine had its own accompanying discriminatory ELISA, but 18% of the virus positive pigs never seroconverted in these tests.

Animals↗

PubMed2003

Protection of pigs against 'in contact' challenge with classical swine fever following oral or subcutaneous vaccination with a recombinant porcine adenovirus.

A recombinant porcine adenovirus expressing the classical swine fever virus (CSFV) gp55 gene (rPAdV-gp55) was administered to commercially available outbred pigs via the subcutaneous or oral route and their susceptibility to 'in contact' challenge with classical swine fever determined. Animals vaccinated subcutaneously with a single dose of recombinant vaccine and challenged by 'in contact' exposure were protected from disease, whereas pigs given an equivalent single oral dose did not survive challenge. However, pigs given two oral doses of rPAdV-gp55, 22 days apart, were completely protected from disease. In addition, two doses of rPAdV-gp55 given subcutaneously was shown to boost CSFV neutralising antibody compared with a single dose, but neither a single dose nor two doses given orally induced detectable neutralising antibody responses.

Adenoviruses, Porcine↗

PubMed1999

Classical swine fever virus is genetically stable in vitro and in vivo.

Phylogenetic analyses of large numbers of classical swine fever strains have revealed a high degree of sequence conservation in the genomic regions examined, suggesting either a recent common ancestor or a low evolution rate. This low variability is in contrast to findings with other RNA viruses. To investigate the consequence of this apparent genetic stability on phylogenetic examinations, the Belgian field isolate Wingene'93 was passaged in pigs as well as in cell culture by various methods. Sequence analyses of viruses collected after various passages in three target regions proposed for phylogenetic studies (5' NTR, E2, and NS5B) revealed a complete sequence conservation. Only when the amount of passaged virus was lowered, mimicking a genetic bottleneck, a single point mutation was observed in the E2 gene. Additionally, only four nucleotide substitutions were observed when the genome of a virus obtained after 96 cell passages in persistently infected cells was compared with its parental virus, the recombinant virus derived from an infectious cDNA clone of CSFV strain Alfort/187. This low mutation frequency observed both in vitro and in vivo demonstrates that classical swine fever virus is genetically stable. Hence, even minor mutations can be considered significant in molecular epidemiological studies.

Animals↗