[Show abstract][Hide abstract] ABSTRACT: African swine fever (ASF) is one of the most complex viral diseases affecting both domestic and wild pigs. It is caused by ASF virus (ASFV), the only DNA virus which can be efficiently transmitted by an arthropod vector, soft ticks of the genus Ornithodoros. These ticks can be part of ASFV-transmission cycles, and in Europe, O. erraticus was shown to be responsible for long-term maintenance of ASFV in Spain and Portugal. In 2014, the disease has been reintroduced into the European Union, affecting domestic pigs and, importantly, also the Eurasian wild boar population. In a first attempt to assess the risk of a tick-wild boar transmission cycle in Central Europe that would further complicate eradication of the disease, over 700 pre-existing serum samples from wild boar hunted in four representative German Federal States were investigated for the presence of antibodies directed against salivary antigen of Ornithodoros erraticus ticks using an indirect ELISA format.
Out of these samples, 16 reacted with moderate to high optical densities that could be indicative of tick bites in sampled wild boar. However, these samples did not show a spatial clustering (they were collected from distant geographical regions) and were of bad quality (hemolysis/impurities). Furthermore, all positive samples came from areas with suboptimal climate for soft ticks. For this reason, false positive reactions are likely.
In conclusion, the study did not provide stringent evidence for soft tick-wild boar contact in the investigated German Federal States and thus, a relevant involvement in the epidemiology of ASF in German wild boar is unlikely. This fact would facilitate the eradication of ASF in the area, although other complex relations (wild boar biology and interactions with domestic pigs) need to be considered.
Full-text · Article · Dec 2016 · BMC Veterinary Research
[Show abstract][Hide abstract] ABSTRACT: Chimeric pestiviruses have shown great potential as marker vaccine candidates against pestiviral infections. Exemplarily, we describe here the construction and testing of the most promising classical swine fever vaccine candidate “CP7_E2alf” in detail. The description is focused on classical cloning technologies in combination with reverse genetics.
[Show abstract][Hide abstract] ABSTRACT: Classical swine fever (CSF) is a viral disease of pigs with tremendous socio-economic impact. In outbreak situations, genetic typing is carried out for the purpose of molecular epidemiology in both domestic pigs and wild boar. These analyses are usually based on harmonized partial sequences. However, for high resolution analyses towards the understanding of genetic variability and virus evolution, full-genome sequences are more appropriate. In this study, a unique set of representative virus strains was investigated that was collected during an outbreak in French free-ranging wild boar in the Vosges-du-Nord Mountains between 2003 and 2007. Comparative sequence and evolutionary analyses of the nearly full-length sequences showed only slow evolution of CSFV strains over the years, and no impact of vaccination on mutation rates. However, substitution rates varied among protein genes and furthermore, a spatial and temporal pattern could be observed whereby two separate clusters were formed that coincided with physical barriers.
No preview · Article · Dec 2015 · Journal of General Virology
[Show abstract][Hide abstract] ABSTRACT: Classical swine fever (CSF) is among the most detrimental diseases for the swine industry worldwide. Infected wild boar populations can play a crucial role in CSF epidemiology and controlling wild reservoirs is of utmost importance for preventing domestic outbreaks. Oral mass vaccination (OMV) has been implemented to control CSF in wild boars and limit the spill over to domestic pigs. This retrospective overview of vaccination experiences illustrates the potential for that option. The C-strain live vaccine was confirmed to be highly efficacious and palatable baits were developed for oral delivery in free ranging wild boars. The first field trials were performed in Germany in the 1990's and allowed deploying oral baits at a large scale. The delivery process was further improved during the 2000's among different European countries. Optimal deployment has to be early regarding disease emergence and correctly designed regarding the landscape structure and the natural food sources that can compete with oral baits. OMV deployment is also highly dependent on a local veterinary support working closely with hunters, wildlife and forestry agencies. Vaccination has been the most efficient strategy for CSF control in free ranging wild boar when vaccination is wide spread and lasting for a sufficient period of time. Alternative disease control strategies such as intensified hunting or creating physical boundaries such as fences have been, in contrast, seldom satisfactory and reliable. However, monitoring outbreaks has been challenging during and after vaccination deployment since OMV results in a low probability to detect virus-positive animals and the live-vaccine currently available does not allow serological differentiation of infected from vaccinated animals. The development of a new marker vaccine and companion test is thus a promising option for better monitoring outbreaks during OMV deployment as well as help to better determine when to stop vaccination efforts. After rabies in red fox, the use of OMV against CSF in European wild boar can be considered as a second example of successful disease control in wildlife. The 30 years of disease control experience included in this review may provide options for improving future disease management within wild populations.
Full-text · Article · Nov 2015 · Frontiers in Microbiology
[Show abstract][Hide abstract] ABSTRACT: RNA viruses have the highest known mutation rates. Consequently it is likely that a high proportion of individual RNA virus genomes, isolated from an infected host, will contain lethal mutations and be non-functional. This is problematic if the aim is to clone and investigate high-fitness, functional cDNAs and may also pose problems for sequence-based analysis of viral evolution. To address these challenges we have performed a study of the evolution of classical swine fever virus (CSFV) using deep sequencing and analysis of 84 full-length cDNA clones, each representing individual genomes from a moderately virulent isolate. In addition to here being used as a model for RNA viruses generally, CSFV has high socioeconomic importance and remains a threat to animal welfare and pig production. We find that the majority of the investigated genomes are non-functional and only 12% produced infectious RNA transcripts. Full length sequencing of cDNA clones and deep sequencing of the parental population identified substitutions important for the observed phenotypes. The investigated cDNA clones were furthermore used as the basis for inferring the sequence of functional viruses. Since each unique clone must necessarily be the descendant of a functional ancestor, we hypothesized that it should be possible to produce functional clones by reconstructing ancestral sequences. To test this we used phylogenetic methods to infer two ancestral sequences, which were then reconstructed as cDNA clones. Viruses rescued from the reconstructed cDNAs were tested in cell culture and pigs. Both reconstructed ancestral genomes proved functional, and displayed distinct phenotypes in vitro and in vivo. We suggest that reconstruction of ancestral viruses is a useful tool for experimental and computational investigations of virulence and viral evolution. Importantly, ancestral reconstruction can be done even on the basis of a set of sequences that all correspond to non-functional variants.
[Show abstract][Hide abstract] ABSTRACT: Question: Domestic pigs and Eurasian wild boar (Sus scrofa) share several important viral and bacterial pathogens. Therefore, direct and indirect contacts between domestic pigs and wild boar present a risk of pathogen spill-over and can lead to long-term perpetuation of infection. Biological indicators could be a powerful tool to understand and characterize contacts between wild boar and domestic pigs. Here, faecal E. coli were explored as potential biological indicator under experimental conditions.
Methods: Within an animal trial domestic pigs (group 2, n=8) were brought into contact with faecal material of wild boar (group 1, n=8). Before and three to five weeks after transmission of faeces fecal samples of both groups were collected, coliforme bacteria isolated, and the bacteria tested by CHEF-PFGE for clonal relatedness. The study was meant as test and calibration phase for potential field studies.
Results: Eighty-eight individual E. coli clones were detected by XbaI restriction and PFGE analysis. Selecting only one isolates representing a distinct clone from an individual faecal sample, 123 E. coli isolates were further analysed. Overall, 17 different clones were found in several animals of a group (1 or 2) or both samples from one animal. Additionally, five clones were detected in group 1 as well as in contact group 2.
Conclusions: The data gained in our pilot study suggest that faecal E. coli can be used as biological indicator of contact between wild boar and domestic pig. Based on these promising results, future field studies will especially target the practicability of E. coli microbiome molecular typing as surrogate of contacts at the
This study is funded by the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement n° 311931 (ASFORCE).
[Show abstract][Hide abstract] ABSTRACT: Recently, CP7_E2alf (Suvaxyn(®)CSF Marker), a live marker vaccine against classical swine fever virus, was licensed through the European Medicines Agency. For application of such a genetically engineered virus under field conditions, knowledge about its genetic stability is essential. Here, we report on stability studies that were conducted to assess and compare the mutation rate of CP7_E2alf in vitro and in vivo. Sequence analyses upon passaging confirmed the high stability of CP7_E2alf, and no recombination events were observed in the experimental setup. The data obtained in this study confirm the genetic stability of CP7_E2alf as an important safety component.
No preview · Article · Sep 2015 · Archives of Virology
[Show abstract][Hide abstract] ABSTRACT: Domestic pigs and Eurasian wild boar (Sus scrofa) share several important viral
and bacterial pathogens. Therefore, direct and indirect contacts between domestic
pigs and wild boar present a risk of pathogen spillover and can lead to long-term
perpetuation of infection. Biological indicators could be a powerful tool to
understand and characterize contacts between wild boar and domestic pigs. Here,
faecal Escherichia coli and Hepatitis E virus (HEV) were explored as potential
biological indicators under experimental conditions. The data gained in our pilot
study suggest that faecal E. coli can be used as biological indicator of contact
between wild boar and domestic pig. For HEV, faecal transmission was also
confirmed. However, molecular studies on full-genome basis did not reveal
markers that would allow tracing of transmission direction. Based on these
promising results, future field studies will especially target the practicability of
E. coli microbiome molecular typing as surrogate of contacts at the wildlife–livestock
[Show abstract][Hide abstract] ABSTRACT: Background:
Over the last years, porcine epidemic diarrhea virus (PEDV) has caused devastating enteric diseases in the US and several countries in Asia, while outbreaks in Europe have only been reported sporadically since the 1980s. At present, only insufficient information is available on currently circulating PEDV strains in Europe and their impact on the European swine industry. In this case report, we present epidemic outbreaks of porcine epidemic diarrhea in three farms in South-Western Germany.
Epidemic outbreaks of diarrhea affecting pigs of all age groups were reported in three farms, one fattening farm and two piglet producing farms, in South-Western Germany between May and November 2014. In the fattening farm yellowish, watery diarrhea without evidence of mucus or blood was associated with a massive reduction of feed consumption. Severity of clinical signs and mortality in young suckling pigs varied significantly between the two affected sow farms. While mortality in suckling piglets reached almost 70 % in one sow herd, no increase in suckling piglet mortality was observed in the second sow farm. In all three cases, PEDV was confirmed in feces and small intestines by RT-qPCR. Phylogenetic analyses based on full-length PEDV genomes revealed high identity among strains from all three herds. Moreover, the German strains showed very high nucleotide identity (99.4 %) with a variant of PEDV (OH851) that was isolated in the United States in January 2014. This strain with insertions and deletions in the S-gene (so called INDEL strains) was reported to show lower virulence. Slightly lower identities were found with other strains from the US and Asia.
Phylogenetic information on the distribution of PEDV strains in Europe is severely lacking. In this case report we demonstrate that acute outbreaks of PEDV occurred in southern Germany in 2014. Current strains were clearly different from isolates found in the 1980s and were closely related to a PEDV variant found in the US in 2014. Moreover, the present case report indicates that variant strains of PEDV, containing insertions and deletions in the S gene, which were reported to be of lower virulence, might be able to cause high mortality in suckling piglets.
Full-text · Article · Jul 2015 · BMC Veterinary Research
[Show abstract][Hide abstract] ABSTRACT: Classical swine fever is a highly contagious disease that affects domestic and wild pigs worldwide. The causative agent of the disease is Classical swine fever virus (CSFV), which belongs to the genus
within the family
. On the genome level, CSFV can be divided into three genotypes with three to four sub-genotypes. Those genotypes can be assigned to distinct geographical regions. Knowledge about CSFV diversity and distribution is important for the understanding of disease dynamics and evolution, and can thus help to design optimized control strategies. For this reason, the geographical pattern of CSFV diversity and distribution are outlined in the presented review. Moreover, current knowledge with regard to genetic virulence markers or determinants and the role of the quasispecies composition is discussed.
No preview · Article · Jun 2015 · Animal Health Research Reviews
[Show abstract][Hide abstract] ABSTRACT: African swine fever (ASF), a disease notifiable to the World Organization of Animal Health (OIE), is characterized by severe, unspecific clinical signs and high mortality rates. Hosts for ASF virus (ASFV) are only members of the family Suidae and soft ticks of the genus Ornithodoros. Currently, no vaccine is available and therefore, the control is primarily based on strict sanitary measures. The most important part is the early detection of the disease within affected animal holdings and the fast and reliable confirmation by laboratory diagnosis. Infections of domestic pigs and European wild boar with recent Armenian, Sardinian, Lithuanian or Kenyan ASFV isolates lead to severe, acute disease courses with the predominant symptom of high fever (> 41 degrees C) accompanied by further unspecific clinical signs such as lethargy, loss of appetite, diarrhoea, respiratory symptoms, and an increased bleeding tendency. In experimental infection studies the mortality rate reached 100%. The most prominent pathomorphological findings included ebony-colored gastrohepatic lymph nodes, lung oedema, petechiae in the renal cortex, and oedema of the gallbladder wall. In the light of the current epidemiological situation with endemic ASFV infections on Sardinia, outbreaks in Russia and several Eastern EU Member States there is a risk for an introduction in further, previously unaffected EU countries including Germany. Hence, appropriate sample materials (serum, blood, spleen) of domestic pigs with unspecific clinical symptoms or pathomorphological findings should be examined for both ASFV and classical swine fever virus.
No preview · Article · May 2015 · Berliner und Münchener tierärztliche Wochenschrift
[Show abstract][Hide abstract] ABSTRACT: In 2007, African swine fever virus (ASFV) was introduced into the Transcaucasian countries and Russia. Since then, it has spread alarmingly and reached the European Union. ASFV strains are highly virulent and lead to almost 100 % mortality under experimental conditions. However, the possibility of dose-dependent disease courses has been discussed. For this reason, a study was undertaken to assess the risk of chronic disease and the establishment of carriers upon low-dose oronasal infection of domestic pigs and European wild boar. It was demonstrated that very low doses of ASFV are sufficient to infect especially weak or runted animals by the oronasal route. Some of these animals did not show clinical signs indicative of ASF, and they developed almost no fever. However, no changes were observed in individual animal regarding the onset, course and outcome of infection as assessed by diagnostic tests. After amplification of ASFV by these animals, pen- and stablemates became infected and developed acute lethal disease with similar characteristics in all animals. Thus, we found no indication of prolonged or chronic individual courses upon low-dose infection in either species. The scattered onset of clinical signs and pathogen detection within and among groups confirms moderate contagiosity that is strongly linked with blood contact. In conclusion, the prolonged course at the "herd level" together with the exceptionally low dose that proved to be sufficient to infect a runted wild boar could be important for disease dynamics in wild-boar populations and in backyard settings.
Full-text · Article · Apr 2015 · Archives of Virology
[Show abstract][Hide abstract] ABSTRACT: Since 2013, highly virulent porcine epidemic diarrhea virus has caused considerable economic losses in the United States. To determine the relation of US strains to those recently causing disease in Germany, we compared genomes and found that the strain from Germany is closely related to variants in the United States.
Full-text · Article · Mar 2015 · Emerging infectious diseases
[Show abstract][Hide abstract] ABSTRACT: Classical swine fever virus (CSFV) is the causative agent of a severe multi-systemic disease of pigs. While several aspects of virus-host-interaction are known, the early steps of infection remain unclear. For the closely related bovine viral diarrhea virus (BVDV), a cellular receptor is known: bovine complement regulatory protein CD46. Given that these two pestiviruses are closely related, porcine CD46 is also a candidate receptor for CSFV. In addition to CD46, cell-culture-adapted CSFV strains have been shown to use heparan sulfates as an additional cellular factor. In the present study, the interaction of field-type and cell-culture-adapted CSFV with a permanent porcine cell line or primary macrophages was assessed using anti-porcine CD46 monoclonal antibodies and a heparan-sulfate-blocking compound, DSTP-27. The influence of receptor blocking was assessed using virus titration and quantitative PCR. Treatment of cells with monoclonal antibodies against porcine CD46 led to a reduction of viral growth in both cell types. The effect was most pronounced with field-type CSFV. The blocking could be enhanced by addition of DSTP-27, especially for cell-culture-adapted CSFV. The combined use of both blocking agents led to a significant reduction of viral growth but was also not able to abolish infection completely. The results obtained in this study showed that both porcine CD46 and heparan sulfates play a major role in the initial steps of CSFV infection. Additional receptors might also play a role for attachment and entry; however, their impact is obviously limited in vitro in comparison to CD46 and heparan sulfates.
No preview · Article · Jan 2015 · Archives of Virology
[Show abstract][Hide abstract] ABSTRACT: Classical swine fever (CSF) is a highly contagious viral disease of pigs with tremendous socio-economic impact. Vaccines are available for disease control. However, most industrialized countries are implementing stamping out strategies to eliminate the disease to avoid trade restrictions. These restrictions could be avoided through the use of marker vaccines such as CP7_E2alf. Marker vaccines have to be accompanied by reliable and robust discriminatory assays. In this context, a multiplex microsphere immunoassay for serological differentiation of the CSFV infected from CP7_E2alf vaccinated animals (DIVA) was developed. To this end, three viral proteins, namely CSFV E2, CSFV E(rns) and BVDV E2, were produced in insect cells using a baculovirus expression system and used as antigens in a microsphere immunoassay, which was further evaluated by testing a large panel of pig sera and compared to a well characterized commercial CSFV E2 Antibody ELISA and a "test" version of an improved CSFV E(rns) antibody ELISA. Under a cut-off median fluorescence intensity value of 5522, the multiplex microsphere immunoassay had a sensitivity of 98.5% and specificity of 98.9% for detection of antibodies against CSFV E2. Both the microsphere immunoassay and the CSFV E(rns) ELISA gave the same results for 155 out of 187 samples (82.8%) for the presence of CSFV E(rns) antibodies. This novel multiplex immunoassay is a valuable tool for measuring and differentiating immune responses to vaccination and/or infection in animals.
Preview · Article · Nov 2014 · Clinical and vaccine Immunology: CVI