[Show abstract][Hide abstract] ABSTRACT: This study investigated the attitudes and beliefs of pig farmers and hunters in Germany, Bulgaria and the western part of the Russian Federation towards reporting suspected cases of African swine fever (ASF). Data were collected using a web-based questionnaire survey targeting pig farmers and hunters in these three study areas. Separate multivariable logistic regression models identified key variables associated with each of the three binary outcome variables whether or not farmers would immediately report suspected cases of ASF, whether or not hunters would submit samples from hunted wild boar for diagnostic testing and whether or not hunters would report wild boar carcasses. The results showed that farmers who would not immediately report suspected cases of ASF are more likely to believe that their reputation in the local community would be adversely affected if they were to report it, that they can control the outbreak themselves without the involvement of veterinary services and that laboratory confirmation would take too long. The modelling also indicated that hunters who did not usually submit samples of their harvested wild boar for ASF diagnosis, and hunters who did not report wild boar carcasses are more likely to justify their behaviour through a lack of awareness of the possibility of reporting. These findings emphasize the need to develop more effective communication strategies targeted at pig farmers and hunters about the disease, its epidemiology, consequences and control methods, to increase the likelihood of early reporting, especially in the Russian Federation where the virus circulates.
Transboundary and Emerging Diseases 08/2014; · 2.10 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: African swine fever (ASF) is a highly lethal viral disease of domestic pigs and wild boar. ASF was introduced into the southern Russian Federation in 2007 and is now reported to be spreading in populations of wild and domestic suids. An endemic situation in the local wild boar population would significantly complicate management of the disease in the livestock population. To date no sound method exists for identifying the characteristic pattern of an endemic situation, which describes infection persisting from generation to generation in the same population. To support urgent management decisions at the wildlife-livestock interface, a new algorithm was constructed to test the hypothesis of an endemic disease situation in wildlife on the basis of case reports. The approach described here uses spatial and temporal associations between observed diagnostic data to discriminate between endemic and non-endemic patterns of case occurrence. The algorithm was validated with data from an epidemiological simulation model and applied to ASF case data from southern Russia. Based on the algorithm and the diagnostic data available, the null hypothesis of an endemic situation of ASF in wild boar of the region was rejected.
Preventive Veterinary Medicine 08/2014; · 2.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Classical swine fever (CSF) is one of the most important viral diseases of pigs. Clinical signs may vary from almost inapparent infection to a hemorrhagic fever like illness. Among the host factors leading to different disease courses are age, breed, and immune status. The aim of this study was to compare host responses of different pig breeds upon infection with a recent moderately virulent CSF virus (CSFV) strain, and to assess their impact on the clinical outcome and the efficiency of immune responses. To this means, two domestic pig types (German Landrace and hybrids), were compared to European wild boar. Along with clinical and pathological assessments and routine virological and serological methods, kinetics of immune-cellular parameters were evaluated.
[Show abstract][Hide abstract] ABSTRACT: African swine fever (ASF) is among the most devastating viral diseases of pigs. In recent years, the disease has spread alarmingly. Despite intensive research activities, promising vaccine candidates are still lacking. For this reason, a study was undertaken to re-assess inactivated ASFV preparations with state-of-the-art adjuvants. Inactivated preparations of ASF virus (ASFV) “Armenia08” were adjuvanted with either Polygen™ or Emulsigen®-D, respectively, and used to immunize six weaner pigs two times with a three-week interval. Six weeks after the first immunization, animals were challenged with the homologues highly virulent ASFV. Although ASFV-specific antibodies were detectable in all but one vaccinated animal prior to challenge, no protective effect of immunization was observed. All animals developed acute-lethal ASF and had to be euthanized within eleven days post challenge. A slightly accelerated clinical course in vaccinees could even indicate an antibody dependent enhancement, which could also influence efficacy of other vaccine approaches.
[Show abstract][Hide abstract] ABSTRACT: African swine fever (ASF) is among the most devastating viral diseases of pigs. In recent years, the disease has spread alarmingly. Despite intensive research activities, promising vaccine candidates are still lacking. For this reason, a study was undertaken to re-assess inactivated ASFV preparations with state-of-the-art adjuvants. Inactivated preparations of ASF virus (ASFV) "Armenia08" were adjuvanted with either Polygen™ or Emulsigen(®)-D, respectively, and used to immunize six weaner pigs two times with a three-week interval. Six weeks after the first immunization, animals were challenged with the homologues highly virulent ASFV. Although ASFV-specific antibodies were detectable in all but one vaccinated animal prior to challenge, no protective effect of immunization was observed. All animals developed acute-lethal ASF and had to be euthanized within eleven days post challenge. A slightly accelerated clinical course in vaccinees could even indicate an antibody dependent enhancement, which could also influence efficacy of other vaccine approaches.
[Show abstract][Hide abstract] ABSTRACT: CP7_E2alf is a promising marker vaccine candidate against classical swine fever (CSF). To better understand the mechanisms of protection, cytokine and isotype-specific antibody profiles were investigated in CP7_E2alf vaccinated pigs before and after challenge with the highly virulent CSFV strain "Koslov" at 14days or 6months post-vaccination. The interference of vaccination with CSFV pathogeny-related cytokine responses, previously described following a moderately virulent challenge, was confirmed. However, the levels of additional cytokines, TNF-α and IL-6, were significantly attenuated by vaccination following highly virulent challenge. This vaccine interference with cytokine response was not dependent on the immunization route or the consequence of competition between vaccine and challenge strain. Interestingly, IFN-γ enhancement and persistent high IgG2 levels suggested an important role of cell-mediated immunity in long-term protection against CSFV induced by CP7_E2alf vaccination. IgA production also revealed a stimulation of mucosal immunity, especially after oral administration of the vaccine.
Research in Veterinary Science 01/2014; · 1.77 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Classical Swine Fever is one of the most important infectious diseases for the pig industry worldwide due to its economic impact. Vaccination is an effective means to control disease, however within the EU its regular use is banned owing to the inability to differentiate infected and vaccinated animals, the so called DIVA principle. This inability complicates monitoring of disease and stops international trade thereby limiting use of the vaccine in many regions. The C-strain vaccine is safe to use and gives good protection. It is licensed for emergency vaccination in the EU in event of an outbreak. Two genetic assays that can distinguish between wild type virus and C-strain vaccines have recently been developed. Here the results from a comparison of these two real-time RT-PCR assays in an interlaboratory exercise are presented. Both assays showed similar performance.
[Show abstract][Hide abstract] ABSTRACT: Dysregulation of cytokine responses plays a major role in the pathogenesis of severe and life-threatening infectious diseases like septicemia or viral hemorrhagic fevers. In pigs, diseases like African and classical swine fever are known to show exaggerated cytokine releases. To study these responses and their impact on disease severity and outcome in detail, reliable, highly specific and sensitive methods are needed. For cytokine research on the molecular level, real-time RT-PCRs have been proven to be suitable. Yet, the currently available and most commonly used SYBR Green I assays or heterogeneous gel-based RT-PCRs for swine show a significant lack of specificity and sensitivity. The latter is however absolutely essential for an accurate quantification of rare cytokine transcripts as well as for detection of small changes in gene expressions. For this reason, a harmonized TaqMan-based triplex real-time RT-PCR protocol for the quantitative detection of normalized gene expression profiles of seven porcine cytokines was designed and validated within the presented study. Cytokines were chosen to represent different immunological pathways and targets known to be involved in the pathogenesis of the above mentioned porcine diseases, namely interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-8, tumor necrosis factor (TNF)-α and interferon (IFN)-α. Beta-Actin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as reference genes for normalization. For absolute quantification a synthetic standard plasmid was constructed comprising all target cytokines and reference genes within a single molecule allowing the generation of positive control RNA. The standard as well as positive RNAs from samples, and additionally more than 400 clinical samples, which were collected from animal trials, were included in the validation process to assess analytical sensitivity and applicability under routine conditions. The resulting assay allows the reliable assessment of gene expression profiles and provides a broad applicability to any kind of immunological research in swine.
PLoS ONE 01/2014; 9(9):e108910. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Oral vaccination against classical swine fever (CSF) is a potent tool to control disease outbreaks in wild boar. So far, vaccination campaigns have been carried out using live attenuated vaccines that do not allow serological differentiation of infected from vaccinated animals (DIVA). Although this drawback is acceptable for wild boar, the use of marker vaccines would facilitate studies on disease and vaccination dynamics. Recently, the CSF marker vaccine candidate CP7_E2alf was assessed for oral immunization under laboratory conditions. Promising results prompted efforts to study the vaccine candidate under field conditions and in bait formulation. In this context, two oral vaccination campaigns were carried out with CP7_E2alf bait vaccines in two areas called ‘faunistic-hunting farms’ in the region of Umbria, Italy. One campaign was conducted using single vaccination, the second with the routinely employed double vaccination strategy. Both campaigns were carried out before concerted hunting actions were performed. Bait uptake, vaccine virus detection and antibody responses were assessed along with inspections upon gutting. As a comparator, seven wild boar were hand-fed with baits under laboratory conditions. In the field, bait uptake ranged from 63.7% to 98.7%, whereas antibody prevalence reached only 33.3–35.1%. The marker serology showed a strong influence of sample quality on the test outcome with a total of 85% of samples being classified correctly. Vaccine virus was not detectable. Under hand feeding conditions, six out of seven wild boar took up at least one bait, and five of them showed detectable antibody levels seven weeks after vaccination. These results were supplemented by stability tests. Appropriate stability of vaccine virus was shown both under field and laboratory conditions. In total, most results were in line with our expectations. However, optimization of the DIVA assay has to be attempted in the future.
[Show abstract][Hide abstract] ABSTRACT: In view of the fact that African swine fever (ASF) was recently introduced into the wild boar population of the European Union and that classical swine fever (CSF) keeps reoccurring, targeted surveillance is of utmost importance for early detection. Introduction of both diseases is usually accompanied by an increased occurrence of animals found dead. Thus, fallen wild boar are the main target for passive surveillance. However, encouraging reporting by hunters and sampling of these animals is difficult. Partly, these problems could be solved by providing a pragmatic sampling approach. For this reason, we assessed the applicability of three different dry/semi-dry blood swabs, namely a cotton swab, a flocked swab, and a forensic livestock swab, for molecular swine fever diagnosis. After nucleic acid extraction using manual and automated systems, routine quantitative real-time polymerase chain reactions (qPCR) were carried out. Results obtained from swabs or their fragments were compared to results generated from EDTA blood.
It was shown that reliable detection of both pathogens was possible by qPCR. Shifts in genome copy numbers were observed, but they did not change the qualitative results. In general, all swabs were suitable, but the forensic swab showed slight advantages, especially in terms of cutting and further storage. Robustness of the method was confirmed by the fact that different extraction methods and protocols as well as storage at room temperature did not have an influence on the final outcome. Taken together, swab samples could be recommended as a pragmatic approach to sample fallen wild boar.
[Show abstract][Hide abstract] ABSTRACT: Classical swine fever (CSF) is among the most important viral disease of domestic and feral pigs and has a serious impact on animal health and pig industry. In most countries with industrialized pig production, prophylactic vaccination against CSF is banned, and all efforts are directed towards eradication of the disease, e.g. by culling of infected herds and animal movement restrictions. Nevertheless, emergency vaccination remains an option to minimize the socio-economic impact of outbreaks. For this application, potent vaccines are needed that allow differentiation of infected from vaccinated animals. Among the promising candidates for next generation marker vaccines is the chimeric pestivirus CP7_E2alf. Efficacy studies are usually carried out using highly virulent CSFV strains of genotype 1 that do not mirror the current field situation where strains of genotype 2 predominate. To prove that CP7_E2alf also protects against these strains, efficacy was assessed after single oral vaccination of wild boar and single intramuscular vaccination of domestic pigs using challenge models with recent CSFV strains and the highly virulent strain "Koslov" (genotype 1.1). It could be demonstrated that CP7_E2alf pilot vaccine batches for intramuscular and oral use were able to protect pigs from challenge infection with a highly virulent CSFV. Moreover, solid protection was also achieved in case of challenge infection with recent field strains of genotypes 2.1 and 2.3. Thus, broad applicability under field conditions can be assumed.
[Show abstract][Hide abstract] ABSTRACT: Classical swine fever virus (CSFV) is a positive-sense RNA virus with a high degree of genetic variability among isolates. High diversity is also found in virulence, with strains covering the complete spectrum from avirulent to highly virulent. The underlying genetic determinants are far from being understood. Since RNA polymerases of RNA viruses lack any proof-reading activity, different genome variations called haplotypes, occur during replication. A set of haplotypes is referred to as a viral quasispecies. Genetic variability can be a fitness advantage through facilitating of a more effective escape from the host immune response. In order to investigate the correlation of quasispecies composition and virulence in vivo, we analyzed next-generation sequencing data of CSFV isolates of varying virulence. Viral samples from pigs infected with the highly virulent isolates "Koslov" and "Brescia" showed higher quasispecies diversity and more nucleotide variability, compared to samples of pigs infected with low and moderately virulent isolates.
[Show abstract][Hide abstract] ABSTRACT: Classical swine fever (CSF), a highly contagious disease of pigs caused by the classical swine fever virus (CSFV), can lead to important economic losses in the pig industry. Numerous CSFV isolates with various degrees of virulence have been isolated worldwide, ranging from low virulent strains that do not result in any apparent clinical signs to highly virulent strains that cause a severe peracute hemorrhagic fever with nearly 100% mortality. Knowledge of the molecular determinants of CSFV virulence is an important issue for effective disease control and development of safe and effective marker vaccines. In this review, the latest studies in the field of CSFV virulence are discussed. The topic of virulence is addressed from different angles; nonconventional approaches like codon pair usage and quasispecies are considered. Future research approaches in the field of CSFV virulence are proposed.
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to evaluate the general characteristics of commercially available enzyme-linked immunosorbent assays (ELISAs) to detect antibody against classical swine fever (CSF), as well as to assess their potential use as accompanying marker tests able to differentiate infected from vaccinated animals (DIVA). The Chekit* CSF-Sero and the HerdChek* CSFV Ab, both of which detect antibodies against the E2 protein of classical swine fever virus (CSFV), had the highest sensitivity. Both tests were practicable and showed good reproducibility. Comparable sensitivity was shown by the Chekit* CSF-Marker, an Erns ELISA. However, this test does not allow differentiation between antibodies directed against ruminant pestiviruses and those against CSFV. Therefore, it is not suitable for use with the chimeric marker vaccines tested. The PrioCHECK CSFV Erns was the only ELISA suitable for use in DIVA with marker vaccines containing Erns proteins from ruminant pestiviruses. However, this test was less sensitive and selective than the E2-ELISAs and cannot be recommended.
Revue scientifique et technique (International Office of Epizootics) 12/2012; 31(3):997-1010. · 0.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Currently no live DIVA (Differentiating Infected from Vaccinated Animals) vaccines against classical swine fever (CSF) are available. The aim of this study was to investigate whether chimeric pestivirus vaccine candidates (CP7_E2alf, Flc11 and Flc9) are able to protect pigs against clinical signs, and to reduce virus shedding and virus transmission, after a challenge with CSF virus (CSFV), 7 or 14 days after a single intramuscular vaccination. In these vaccine candidates, either the E2 or the E(rns) encoding genome region of a bovine viral diarrhoea virus strain were combined with a cDNA copy of CSFV or vice versa. Furthermore, currently available serological DIVA tests were evaluated. The vaccine candidates were compared to the C-strain. All vaccine candidates protected against clinical signs. No transmission to contact pigs was detected in the groups vaccinated with C-strain, CP7_E2alf and Flc11. Limited transmission occurred in the groups vaccinated with Flc9. All vaccine candidates would be suitable to stop on-going transmission of CSFV. For Flc11, no reliable differentiation was possible with the current E(rns)-based DIVA test. For CP7_E2alf, the distribution of the inhibition percentages was such that up to 5% false positive results may be obtained in a large vaccinated population. For Flc9 vaccinated pigs, the E2 ELISA performed very well, with an expected 0.04% false positive results in a large vaccinated population. Both CP7_E2alf and Flc9 are promising candidates to be used as live attenuated marker vaccines against CSF, with protection the best feature of CP7_E2alf, and the DIVA principle the best feature of Flc9.
[Show abstract][Hide abstract] ABSTRACT: African swine fever (ASF) is among the most important viral diseases that can affect domestic and feral pigs. Both clinical signs and pathomorphological changes vary considerably depending on strain virulence and host factors. Acute infections with highly virulent virus strains lead to a clinical course that resembles a viral haemorrhagic fever that is characterized by pronounced depletion of lymphoid tissues, apoptosis of lymphocyte subsets, and impairment of haemostasis and immune functions. It is generally accepted that most lesions can be attributed to cytokine-mediated interactions triggered by infected and activated monocytes and macrophages, rather than by virus-induced direct cell damage. Nevertheless, most pathogenetic mechanisms are far from being understood. This review summarizes the current knowledge and discusses implications and research gaps.
[Show abstract][Hide abstract] ABSTRACT: Classical swine fever (CSF) is a multi-systemic disease that can be accompanied by severe haemorrhagic lesions. The underlying pathogenetic mechanisms are still far from being understood, though disseminated intravascular coagulation (DIC) was discussed as a major factor. In the presented study, the direct thrombin inhibitor hirudin was used in an attempt to elucidate the role of the coagulation system in the pathogenesis of CSF-induced haemorrhagic lesions. Two groups of piglets (n=5) were infected with highly virulent CSF virus (CSFV) strain CSF0634. One group underwent daily treatment with hirudin, the other served as untreated challenge infection control. Assessment of clinical signs using a clinical score system, coagulation tests, and blood counts were performed daily. Both groups developed acute-lethal CSF with haemorrhagic lesions. Although changes in the coagulation system were seen in the late stages of CSFV infection, our results strongly suggest that DIC does not present the crucial event in the pathogenesis of haemorrhagic lesions.
[Show abstract][Hide abstract] ABSTRACT: Marker vaccines offer the possibility to differentiate classical swine fever (CSF) infected from CSF vaccinated animals based on serology and their implementation will ensure free trade with pigs. Therefore, new generations of promising marker vaccines have been developed, among them the chimeric vaccine CP7_E2alf. However, in populations previously vaccinated with live attenuated vaccines like the C-strain, passive immunity through maternal antibodies can interfere with efficacy of CP7_E2alf vaccination. Therefore, the efficacy of CP7_E2alf was examined in piglets from sows vaccinated once intramuscularly with C-strain vaccine 4weeks before farrowing. Thus, these piglets were vaccinated intramuscularly with CP7_E2alf at the age of 5 or 8weeks. Subsequently, the piglets and their mock-vaccinated littermate controls were challenged 2weeks post vaccination with highly virulent Classical swine fever virus (CSFV) strain "Koslov". CP7_E2alf provided clinical protection upon challenge as no severe clinical signs or mortality was observed in the vaccinated piglets. Post mortem examination revealed pathological changes associated to CSFV only in the mock-vaccinated piglets. No infectious CSFV could be isolated from the tonsils of the vaccinated piglets. Two weeks after vaccination at the time of challenge, the vaccinated piglets only, had an increase in the ELISA antibody titer. Interestingly, the maternally derived immunity in the mock-vaccinated control piglets seems to neutralize the challenge virus. Thus, the previously observed 100% mortality in naïve (negative for antibodies to CSFV) piglets infected with CSFV Koslov was reduced in the control piglets of this study to 30% for challenge at the age of 7weeks and 50% at the age of 10weeks, respectively. In conclusion, CP7_E2alf proved to be effective in preventing mortality, severe clinical signs and pathological lesions in 5 or 8weeks old piglets positive for maternal antibodies derived from sows vaccinated intramuscularly 4weeks before farrowing with one dose of C-strain vaccine.
[Show abstract][Hide abstract] ABSTRACT: Classical swine fever virus (CSFV) C-strain "Riems" escape variants generated under selective antibody pressure with monoclonal antibodies and a peptide-specific antiserum in cell culture were investigated. Candidates with up to three amino acid exchanges in the immunodominant and highly conserved linear TAV-epitope of the E2-glycoprotein, and additional mutations in the envelope proteins ERNS and E1, were characterized both in vitro and in vivo.It was further demonstrated, that intramuscular immunization of weaner pigs with variants selected after a series of passages elicited full protection against lethal CSFV challenge infection. These novel CSFV C-strain variants with exchanges in the TAV-epitope present potential marker vaccine candidates. The DIVA (differentiating infected from vaccinated animals) principle was tested for those variants using commercially available E2 antibody detection ELISA. Moreover, direct virus differentiation is possible using a real-time RT-PCR system specific for the new C-strain virus escape variants or using differential immunofluorescence staining.
Veterinary Research 04/2012; 43(1):33. · 3.43 Impact Factor