Molecular epidemiology of classical swine fever in Italy

To gain an insight into the molecular epidemiology of classical swine fever (CSF) in Italy, virus isolates originating from outbreaks that occurred between 1985 and 2000 in wild boar or in domestic pigs in mainland Italy and in Sardinia were analysed by genetic typing. For this, a fragment (190 nucleotides) of the E2 glycoprotein gene was sequenced and phylogenetic analyses were performed, including older Italian isolates and isolates from recent outbreaks in Europe for comparison. The results show that in mainland Italy, several independent epidemiological events occurred in the last decade. In the north of the country, viruses of genotype 2.2 have persisted in wild boar, causing sporadic outbreaks in domestic pigs. In contrast, viruses of subgroups 2.1 and 2.3 appeared only intermittently in different regions of the mainland. In 1997, classical swine fever virus (CSFV) isolates belonging to the subgroup 2.1 and genetically and epidemiologically related to the Dutch isolate in Venhorst, affected domestic pigs exclusively. The isolates of subgroup 2.3, derived from wild boar as well as from domestic pigs were closely related to isolates from Germany and Poland. In Sardinia, CSF is an endemic in wild boar and affects domestic pigs also. Genetic typing showed that viruses of subgroups 1.1 and 2.3 have been present, the last ones being unrelated to the mainland viruses of the same subgroup. Due to the large quantities of pig and wild boar meat imported in some parts of Italy, it cannot be established if these viruses were always present in either the mainland or Sardinia, or if they represent recent introductions.     

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.     

Case report: the significance of genotyping for the epidemiological tracing of classical swine fever (CSF)

In Germany, eleven outbreaks of CSF in domestic pig holdings were reported in 2002. They occurred exclusively in regions where CSF virus circulated in the wild boar population. In ten cases the phylogenetic analysis revealed that the isolates from domestic pigs and wild boar had identical sequences in the 5' non-translated region (5'NTR). However, in one case a subtype was isolated which was slightly different from the virus subtype found in the wild boar population of that region. This case is decribed in detail. The epidemiological significance of different diagnostic methods is discussed, in particular the genetic typing of CSF virus isolates.     

Genetic typing of classical swine fever viruses--a review

Classical swine fever (CSF) is a notifiable disease of domestic pigs and wild boar. It is caused by the highly contagious CSF virus and in its acute form the disease generally results in high morbidity and mortality. Due to the great economical impact an outbreak can cause to the pig industry it is one of the most important swine diseases worldwide. To limit the damage in the case of a new outbreak it is necessary to identify the virus as fast as possible. This information helps epidemiologists to trace the origin of the virus and to follow the virus spread. Genetic typing revealed that CSF virus genotypes, subgroups and types show a regional distribution making it an important tool for epidemiologists. Meanwhile, besides epidemiological data and nucleotide sequences from European isolates, information from isolates from South- and Central America, the Caribbean, Asia and recently from South Africa have become available. The data are stored in a database in the EU Reference Laboratory for CSF, accessible by the WWW (http://viro08.tiho-hanno ver.de). A new module was implemented that allows efficient automated genotyping.     

Classical swine fever--an update

Classical swine fever (CSF) is a serious and contagious viral disease of pigs and wild boar with a widespread worldwide distribution. The immunopathology of the disease is poorly understood, but the ability of the CSF virus to infect cells without triggering apoptosis and to kill uninfected cells is probably highly significant. The virus may be spread by various direct and indirect methods, but in most cases the exact mechanisms involved in local spread between farms are not known. Excellent diagnostic tools and typing methods are available, but tests that could be performed on-farm, in pre-clinically infected pigs or on meat would also be advantageous. A more complete picture of the viruses circulating in different parts of the world is needed. There is great interest to develop and use marker vaccines for the control of CSF in domestic pigs and in wild boar. Epidemiological modelling is increasingly used to evaluate control options.     

Genetic typing of German isolates of classical swine fever virus]

During the last decade several outbreaks of classical swine fever (CSF) occurred in Germany in domestic pigs and in wild boar, respectively. Two major epidemics which also affected other EU Member States were recorded. To support epidemiological investigations genetic typing was applied and virus isolates originating from different outbreaks in Germany were assigned to groups and virus types. Two genomic regions were selected for the phylogenetic analysis, namely 150 nucleotides from the 5' non-translated region (5'-NTR) and 190 nucleotides from the E2 glycoprotein gene. All German CSF virus isolates of the nineties (Group 2) were distinct from former reference strains (Group 1). Within Group 2 both genomic regions allowed to distinguish three subgroups, namely 2.1, 2.2 and 2.3. Within subgroup 2.3 five virus types could be discriminated using the 5'-NTR sequences. These are 2.3*Uelzen and 2.3*Spreda, mainly with isolates from Lower Saxony, as well as 2.3*Rostock, 2.3*Güstrow and 2.3*Spante, mainly with isolates from Eastern Germany. Analysis of the E2 gene fragment allowed a better discrimination between single isolates, but only two virus types could be defined: 2.3*MV/BB, comprising the isolates from Eastern Germany, and 2.3*NI, with the isolates from Lower Saxony. Genetic typing allowed to discriminate between isolates involved in different CSF epidemics, and was useful for tracing the origin and spread of CSF viruses. Due to the close relationship between German CSF virus isolates, epidemiological data are a prerequisite for the interpretation of the results obtained by genetic typing. In addition, at least both genomic regions suggested here should be analysed to determine the identity of a new isolate.     

Emergence of 2.1. subgenotype of classical swine fever virus in pig population of India in 2011

Background: Limited studies are available on molecular epidemiology of classical swine fever virus (CSFV) in India and are restricted to domestic pigs. These studies show the presence of 1.1. genotype.

Hypothesis/objectives: The aim of the present study was to subgenotype four CSFV isolates, two each from the outbreaks of CSF in wild (Sus scrofa) and domestic pigs of Mizoram state, India, in 2011.

Animals and methods: CSFV isolates were subjected to nucleotide sequencing in E2 and NS5B genomic regions. Phylogenetic analysis of the isolates in both genomic regions was carried out with 39 Indian isolates (4 isolates from the present study of Mizoram state and 35 isolates from the other states of India) and 57 reference sequences retrieved from the GenBank database. Two of the 39 isolates from India were collected from wild boar and were subgenotyped as 2.1. Out of 37 isolates from domestic pigs, only two were subgenotyped as 2.1.

Results: The analysis revealed the emergence of 2.1. subgenotype of CSFV in both wild and domestic pigs in India.

Conclusions and clinical importance: The isolates from domestic pigs of Mizoram state (CSF/MZ/KOL/73 and CSF/MZ/AIZ/115) were grouped in genotype 1 and subgenotype 1.1., thus confirming that the source of CSF outbreaks in domesticated pigs in Mizoram was not from wild pigs. The current study forms an essential step for better understanding of the epidemiology of 2.1 subgroup as well as the movement and spread of the disease in India.     

Efficacy of lyophilised C-strain vaccine after oral immunisation of domestic pigs and wild boar against classical swine fever: first results

The aim of this study was to evaluate the efficacy of lyophilised C-strain vaccine in domestic pigs and wild boar after oral application. A new spherical bait form (diameter 3 cm) containing lyophilised vaccine virus and the recent vaccine baits were used for animal experiments. Four vaccination groups were established in experiment 1 (group 1: recent liquid bait vaccine; group 2: spherical baits containing one dose of the lyophilised vaccine; groups 3 (domestic pigs) and 4 (wild boar): spherical baits containing two doses of the lyophilised vaccine) and two groups in experiment 2 (group 1: recent liquid bait vaccine; group 2: spherical baits with two doses of the lyophilised vaccine). Challenge was carried out with the highly virulent virus strain "Alfort 187" (using 100 TCID50 in the first and 1.000 TCID50 in the second experiment). Our results showed that the animals vaccinated with lyophilised C-strain vaccine developed high neutralising antibody titres comparable to those obtained after vaccination with the recent bait vaccine. All pigs which picked up the baits remained healthy after challenge. Neither clinical symptoms nor viremia or virus shedding were observed after infection except in one pig (group 2, experiment 2) which had not consumed the vaccine bait. The surviving domestic pigs and wild boar were tested negative for CSFV and viral RNA at the end of the study. This result demonstrates that lyophilised vaccine may become an effective vaccine formulation for oral immunisation of wild boar against CSF in the near future.     

Genetic typing of recent classical swine fever virus isolates from Croatia

During a period of 5 years (1997-2001) several outbreaks of classical swine fever (CSF) were recorded in Croatia. For genetic typing, fragments of 150 nucleotides within the 5'-non-translated region (5'-NTR) and 190 nucleotides within the E2 glycoprotein coding gene of nine field isolates that were derived from domestic pigs and wild boars were used. For better epizootiological understanding, isolates from other European countries were included in the study. The results show that the isolates belong to subgroups 2.1 and 2.3 of CSF virus. Isolates from subgroup 2.1 were collected from domestic pigs during sporadic outbreaks in June 1997 and are genetically closely related. A genomic similarity between these isolates and CSF virus isolates from pigs in other European countries from the same year could also be confirmed. In contrast, the isolate from October 1997 was found to be a member of subgroup 2.3, and is closely related to European CSF virus isolates from outbreaks in the last decade in Western and Central European countries. These results show that two different sources of CSF virus caused outbreaks in Croatia during the same year. Furthermore, a close relationship was found between an isolate from a domestic pig in 1999 and isolates of subgroup 2.3 that originated from Croatian wild boars.     

Detection and analysis of porcine circovirus type 1 in Hungarian wild boars: short communication

Porcine circovirus type 1 (PCV1) is considered to be a non-pathogenic virus detected in cell cultures, vaccines or products used for cell culture preparations, all of them of porcine origin. Serological evidence and genetic studies suggested that PCV1 was widespread in domestic pigs. The presence of PCV1 in wild boars in Germany was also described using serological methods. This paper reports the first detection of PCV1 in Hungarian wild boars. Samples were collected at slaughterhouses and processed for polymerase chain reactions. The complete genome of PCV1 detected in the samples was determined and compared with the available PCV1 sequences of the GenBank database. The genomes formed two distinct clusters with minimum differences, where the Hungarian wild boar PCV1 (WB-H8) grouped together with genomes originating from domestic swine from China and Australia and with a genome detected in a porcine pepsin product.     

Monitoring of classical swine fever in wild boar (Sus scrofa) in Slovenia

Classical swine fever (CSF) is a highly contagious multi-systemic haemorrhagic viral disease of pigs. Not only domestic pigs, but also wild boar appear to play a crucial role in the epidemiology of CSF. Spleen (n = 739) and blood coagulum (n = 562) sampled from wild boars (Sus scrofa) shot in 2002, and serum samples from 746 wild boar shot in 2003 and 2004, were tested throughout Slovenia. In 2002, 17 samples were positive on enzyme-linked immunosorbent assay (ELISA) test for antibodies against classical swine fever virus (CSFV). Positive ELISA test was confirmed by a virus neutralization test. All other samples were negative. This is the first report that describes the epidemiology of CSFV from 2002 on, and the monitoring of the wild boar population in Slovenia at present.     

Emergence of classical swine fever virus in Israel in 2009

Classical swine fever (CSF) re-emerged in Israel in February 2009 after an absence of 62 years. The outbreak occurred on a domestic pig farm in northern Israel and affected domestic pigs and wild boar. On the basis of phylogenetic analysis of a 190 base pair fragment of the E2 glycoprotein gene, the Israeli CSF virus strain belonged to genotype 2.1 and was genetically most similar to a Chinese CSF virus strain.     

Epidemiology of classical swine fever in Germany in the 1990s

In Germany, 424 outbreaks of CSF in domestic pigs and a great number of cases in wild boar were recorded between 1990 and 1998. Most of the federal states ('Bundesl?nder') were affected. Epidemiological data from field investigations combined with genetic typing allowed to distinguish seven unrelated epidemics and a number of sporadic outbreaks in domestic pigs. Detailed epidemiological data was available for 327 outbreaks. It was found that 28% of these were primary outbreaks. Most of them were due to indirect or direct contact to wild boar infected with CSF virus or swill feeding. Infected wild boar remain the main risk for domestic pigs. The most frequent sources of infection in secondary or follow up outbreaks were the trade with infected pigs, neighbourhood contacts to infected farms and other contacts via contaminated persons and vehicles, respectively. An increased risk of virus transmission from infected herds to neighbourhood farms was observed up to a radius of approximately 500m. More than two thirds of the infected herds were discovered due to clinical signs. About 20% were identified by epidemiological tracing on and back. These were scrutinised because contacts to infected herds were evident. In conclusion, tracing of contact herds and clinical examination combined with carefully targeted virological testing of suspicious animals is likely to be the most important measure to immediately uncover secondary outbreaks. Obligatory serological screening in the surveillance and the restriction zones do not seem to be efficient measures to detect follow-up outbreaks.     

Introduction to classical swine fever: virus, disease and control policy

Classical swine fever virus is a spherical enveloped particle of about 40-60 nm in diameter with a single stranded RNA genome of about 12,300 bases with positive polarity, classified as a pestivirus within the family Flaviviridae. Natural hosts are domestic and wild pigs. The virus causes one of the most severe diseases in pigs world wide with grave economic consequences. The clinical picture of classical swine fever is variable, depending on the age of the affected animals and viral virulence. The virus is well characterised and reliable laboratory diagnostic procedures are available. In many parts of the world live attenuated vaccines are being used as a safe and efficient prophylactic tool. However, in EU Member States and several other countries vaccination is prohibited and CSF is controlled by a strict stamping out policy. In order to overcome the disadvantages of conventional vaccination inactivated marker vaccines have been developed that enable the distinction between vaccinated and infected animals. Whether these vaccines will be accepted as an additional tool in the framework of the stamping out policy is not yet decided.     

Simulating the spread of classical swine fever virus between a hypothetical wild-boar population and domestic pig herds in Denmark

Denmark has no free-range wild-boar population. However, Danish wildlife organizations have suggested that wild boar should be reintroduced into the wild to broaden national biodiversity. Danish pig farmers fear that this would lead to a higher risk of introduction of classical swine fever virus (CSFV), which could have enormous consequences in terms of loss of pork exports. We conducted a risk assessment to address the additional risk of introducing and spreading CSFV due to the reintroduction of wild boar. In this paper, we present the part of the risk assessment that deals with the spread of CSFV between the hypothetical wild-boar population and the domestic population. Furthermore, the economic impact is assessed taking the perspective of the Danish national budget and the Danish pig industry. We used InterSpreadPlus to model the differential classical swine fever (CSF) risk due to wild boar. Nine scenarios were run to elucidate the effect of: (a) presence of wild boar (yes/no), (b) locations for the index case (domestic pig herd/wild-boar group), (c) type of control strategy for wild boar (hunting/vaccination) and (d) presence of free-range domestic pigs. The presence of free-range wild boar was simulated in two large forests using data from wildlife studies and Danish habitat data. For each scenario, we estimated (1) the control costs borne by the veterinary authorities, (2) the control-related costs to farmers and (3) the loss of exports associated with an epidemic. Our simulations predict that CSFV will be transmitted from the domestic pig population to wild boar if the infected domestic pig herd is located close to an area with wild boar (<5 km). If an outbreak begins in the wild-boar population, the epidemic will last longer and will occasionally lead to several epidemics because of periodic transfer of virus from groups of infected wild boar to domestic pig herds. The size and duration of the epidemic will be reduced if there are no free-range domestic pig herds in the area with CSF-infected wild boar. The economic calculations showed that the total national costs for Denmark (i.e. the direct costs to the national budget and the costs to the pig industry) related to an outbreak of CSF in Denmark will be highly driven by the reactions of the export markets and in particular of the non-EU markets. Unfortunately, there is a substantial amount of uncertainty surrounding this issue. If hunting is used as a control measure, the average expenses related to a CSF outbreak will be 40% higher if wild boar are present compared with not present. However, a vaccination strategy for wild boar will double the total costs compared with a hunting strategy.     

Experimental infection of wild boar and domestic pigs with a foot and mouth disease virus strain detected in the southeast of Bulgaria in December of 2010

Foot and mouth disease (FMD) was detected in a wild boar in Southeastern Bulgaria in December 2010. The occurrence and spread of the disease in wild cloven-hoofed animals may pose an unexpected and significant threat to FMD virus (FMDV)-free areas within and outside the European Union. So far, only one well documented experimental infection with FMD in wild boar has been published. In order to obtain more epidemiologically relevant data regarding the disease in wild boar we conducted an experiment with the 2010 Bulgarian FMDV type O isolate. Two young wild boar were challenged while two domestic pigs and two additional wild boar served as contact controls. While the domestic pigs developed severe clinical signs of FMD, the wild boar showed relatively mild course of the disease. Viremia started in contact wild boar 2 days post exposure (DPE) and lasted until 6 DPE. The virus shedding lasted until 9 DPE. On 27 DPE, when the animals were slaughtered, viral RNA was detected in lymphoid tissues and oropharyngeal fluid but no virus could be isolated. Commercial ELISAs and virus neutralisation tests detected antibodies against FMDV on 8 or 6 DPE, respectively. The data of the present study will help to understand FMD in wild boar populations and can be used in models to evaluate the potential role of wild boar in FMD epidemiology.     

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.     

The potential negative impacts of the classical swine fever virus on wild boar population in Gifu prefecture,Japan

There is a possibility that classical swine fever (CSF) virus outbreak has negative impacts on wild boar. To adequately manage native wild boar populations, wildlife managers need to gather the field data on wild boar and implement population management practices. We aimed to report the relative abundance index of wild boar before and after this outbreak. Our results showed that relative abundance index declined from 2017 (8.88 wild boars/100 trap days) to 2019 (2.03 wild boars/100 trap days), because of the negative impact of this virus and continuous culling programs. Although the eradication risk from the synergistic effect is low, wildlife managers need to consider the relationship between the trade-off between the risk of CSF and the conservation ecology risk of native species eradication.     

Disease severity declines over time after a wild boar population has been affected by classical swine fever--legend or actual epidemiological process?

Classical swine fever (CSF) is a severe multi-systemic disease that can affect both domestic pigs and wild boar. Past outbreaks in European wild boar involved high-virulent CSF virus (CSFV) strains and were mostly self-limiting. In these cases, morbidity and mortality rates were high in the affected regions. In contrast, endemic infections have been observed in several European wild boar populations in recent decades. Morbidity and mortality rates were much lower despite the fact that outbreaks were still detected via diseased or fallen animals. The virus strains involved were mostly classified as genotype 2.3 strains of moderate virulence causing age-dependent disease outcomes. The mechanisms leading to the establishment and perpetuation of endemicity are still not fully understood, but the factor "moderate virulence" seems to be of considerable importance. In this study, we aim to clarify whether the perception of declined 'CSF severity' could hypothetically reflect the adaptation of an initially high-virulent virus or whether this might be better explained as a misinterpretation of observations. A mechanistic eco-epidemiological model was employed to follow up a highly virulent strain of CSFV introduced into large connected wild boar populations. In the model, the virulence of the CSF virus is represented by case mortality and life expectancy after lethal infection. Allowing for small stochastic variation, these two characteristics of the virus are passed on with every new simulated infection that occurs. Model analysis revealed a decrease from high to moderate case mortality within a few years of simulated perpetuation of the virus. The resulting mortality corresponded to the level where the population average of the infectious period and the basic reproduction number of the disease were maximal. This shift in virulence was sufficient to prolong virus circulation considerably beyond the epidemic phase of the simulated outbreaks. Alternative mechanistic explanations for the decrease in disease severity in a CSF-affected wild boar population were evaluated in the light of the simulation experiments and the available epidemiological or virological evidence. In conclusion, the current virus isolates of subgroup 2.3 might be the ideally adapted variants of the CSF virus for long-term perpetuation in wildlife and indeed may have evolved (once) during past outbreaks in large populations. A repeated perception of a declining severity of disease pattern during the course of a CSF outbreak, however, favours the explanation based on monitoring and detection biases rather than repeated observation of selection against highly virulent virus during the time of virus perpetuation.