Evaluation of blowfly larvae (Diptera: Calliphoridae) as possible reservoirs and mechanical vectors of African swine fever virus.

In 2014, highly virulent African swine fever virus (ASFV) was introduced into the Baltic States and Poland, with new cases being reported almost every week from wild boar and also from domestic pigs. Contrary to initial predictions that the disease would either die out due to the high virulence of the virus strain or spread rapidly in westerly direction, the infection became endemic and spread slowly. The unexpected disease epidemiology led to the hypothesis that hitherto unconsidered factors might contribute to virus persistence and dispersal. To check whether arthropod species feeding and developing on infected carcasses might be involved, larvae of two commonly found blowfly species, Lucilia sericata and Calliphora vicina, were experimentally bred on ASFV-infected spleen tissue. After different time intervals, developing larvae and pupae were tested for infectious virus and viral DNA. By qPCR, contamination of the blowfly larvae and pupae with ASFV-DNA could be demonstrated even after several washing steps, proving the uptake of virus during feeding in the larval stage. However, infectious virus could never be isolated. By contrast, the larvae appeared to have inactivated ASFV in the offered tissue, which might be explained by the known anti-biotic effect of salivary secretions. It is concluded that immature blowfly stages do not play a relevant role as reservoirs or mechanical vectors of ASFV.

Urgent request on avian influenza.

Highly pathogenic avian influenza (HPAI) H5N8 is currently causing an epizootic in Europe, infecting many poultry holdings as well as captive and wild bird species in more than 10 countries. Given the clear clinical manifestation, passive surveillance is considered the most effective means of detecting infected wild and domestic birds. Testing samples from new species and non-previously reported areas is key to determine the geographic spread of HPAIV H5N8 2016 in wild birds. Testing limited numbers of dead wild birds in previously reported areas is useful when it is relevant to know whether the virus is still present in the area or not, e.g. before restrictive measures in poultry are to be lifted. To prevent introduction of HPAIV from wild birds into poultry, strict biosecurity implemented and maintained by the poultry farmers is the most important measure. Providing holding-specific biosecurity guidance is strongly recommended as it is expected to have a high impact on the achieved biosecurity level of the holding. This is preferably done during peace time to increase preparedness for future outbreaks. The location and size of control and in particular monitoring areas for poultry associated with positive wild bird findings are best based on knowledge of the wider habitat and flight distance of the affected wild bird species. It is recommended to increase awareness among poultry farmers in these established areas in order to enhance passive surveillance and to implement enhanced biosecurity measures including poultry confinement. There is no scientific evidence suggesting a different effectiveness of the protection measures on the introduction into poultry holdings and subsequent spread of HPAIV when applied to H5N8, H5N1 or other notifiable HPAI viruses.

Classical Swine Fever in Wild Hog: Report of its Prevalence in Northeast India.

Classical swine fever virus (CSFV) is the causative agent of a highly contagious disease, hog cholera in pigs. The disease is endemic in many parts of the world and vaccination is the only way to protect the animals from CSFV infection. Wild hogs belong to the species Sus Scrofa Cristatus under the family Suidae are quite susceptible to CSFV infection. The epidemiological role concerning classical swine fever (CSF) in India is largely unknown. We report here the three isolated cases of CSF in wild hogs from three National parks, namely Kaziranga National Park, Manas National Park and Jaldapara National Park, from north-east part of India. The post-mortem and histopathological findings were clearly indicative for CSFV infection. The presence of CSFV genome was demonstrated in several organs and tissues collected from hogs died due to viral infection. In addition, CSF-specific antibodies were detected in two wild hogs as well as in eighteen feral pigs from the same locations. The phylogenetic analysis of the partial E2 protein gene and 5' untranslated region of CSFV isolates from the wild hog showed identities with genotype 2.2 of the Indian isolates. Occurrence of CSF in wild hogs may pose a potent threat in the epidemiology of the virus in Northeast part of India. To the best of our knowledge, the report presented in the manuscript is the first comprehensive report on CSF in wild hogs form Northeast India. The findings reported would help us to understand the epidemiology and biology of CSFV in wild animals.

Assessing the potential spread and maintenance of foot-and-mouth disease virus infection in wild ungulates: general principles and application to a specific scenario in Thrace.

Foot-and-mouth disease (FMD), due to infection with serotype O virus, occurred in wild boar and within eleven outbreaks in domestic livestock in the south-east of Bulgaria, Thrace region, in 2011. Hence, the issue of the potential for the spread and maintenance of FMD virus (FMDV) infection in a population of wild ungulates became important. This assessment focused on the spread and maintenance of FMDV infection within a hypothetical wild boar and deer population in an environment, which is characterized by a climate transitional between Mediterranean and continental and variable wildlife population densities. The assessment was based on three aspects: (i) a systematic review of the literature focusing on experimental infection studies to identify the parameters describing the duration of FMDV infection in deer and wild boar, as well as observational studies assessing the occurrence of FMDV infection in wild deer and wild boar populations, (ii) prevalence survey data of wild boar and deer in Bulgaria and Turkey and (iii) an epidemiological model, simulating the host-to-host spread of FMDV infections. It is concluded, based on all three aspects, that the wildlife population in Thrace, and so wildlife populations in similar ecological settings, are probably not able to maintain FMD in the long term in the absence of FMDV infection in the domestic host population. However, limited spread of FMDV infection in time and space in the wildlife populations can occur. If there is a continued cross-over of FMDV between domestic and wildlife populations or a higher population density, virus circulation may be prolonged.

Trapping as an alternative method of eradicating classical swine fever in a wild boar population in Bulgaria.

Between August and November 2009, eight cases of classical swine fever (CSF) occurred in young wild boar in a 25-km2 oak forest3 km south of the river Danube in the north-eastern part of Bulgaria. The wild boar population within the affected area was estimated to be 156 animals, or approximately six boar per km2. To control and eradicate the disease, and in addition to vaccination and hunting, trapping was used to reduce the boar population to below two animals per km2. In total, 124 wild boar were removed from the infected area within three months. Of these, 119 were trapped. In this paper, the authors present trapping as a successful tool to eradicate CSF from an area where hunting and vaccination alone might not be sufficient. Up to seven wild boar could be trapped in a single trap. Furthermore, the spread of CSF virus to the local domestic pig population and to wild boar in neighbouring areas was prevented. By decreasing the wild boar population to fewer than two animals per km2, it was assumed that the virus would no longer circulate and the disease would fade out. In fact, no further CSF cases were diagnosed afterwards. Under Bulgarian and similar conditions, trapping seems to be a more reliable method than hunting for reducing a wild boar population within a short period of time. Furthermore, trapping may be used alone or in combination with hunting, depending on the situation.

Inactivation of classical swine fever virus in porcine casing preserved in salt.

Pig intestines used for the production of natural sausage casings may carry classical swine fever (CSF) virus. Feeding pigs with human food waste that contains pig casings may then spread the virus to CSF-free animals. Casings derived from a pig experimentally infected with CSF by dosing with 10(6) tissue culture infectious doses (TCID50) of the highly virulent CSF virus strain "Koslov", were treated with phosphate supplemented or citrate supplemented NaCl, instead of with NaCl alone, which is the standard preservation treatment for casings. Treated casings were stored for 30 days at either 4 degrees C or 20 degrees C. After storage the casings were fed to 16 susceptible pigs. CSF infection was confirmed in the four animals that had been fed casings treated with citrate supplemented salt and stored at 4 degrees C. All other animals remained healthy. It is therefore possible to avoid the inadvertent spread of CSF virus via porcine sausage casings by treating casings with phosphate supplemented salt and storing them for 30 days at temperatures over 4 degrees C.

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

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

A simple statistical approach for assessing inter-laboratory comparison tests for classical swine fever diagnosis.

The aim of this study was to compare on an objective basis the results obtained during five classical swine fever (CSF) ring tests conducted in Germany between 1999 and 2003. A novel and simple statistical approach used in behavioural sciences was used. For each ring test, the regional laboratories received a panel of five lyophilized pig sera. The panel contained CSF virus positive and negative samples. The final task of the laboratory was to ascertain if a serum sample was positive for CSF or not. Some sera were very easy to diagnose as CSF positive while some sera had border line values and proved to be challenging. Depending on the degree of difficulty the sera were divided into five categories. The evaluation of the ring test results was performed using a scoring system based on a score from -3 to +3 which takes into consideration the degree of difficulty to produce a correct diagnosis. To compare the results between different laboratories and/or between different ring tests more easily the total score of one laboratory was expressed in percentage. The final analysis of the data showed that the CSF diagnostic quality improved continuously.

Diagnostic evaluation of a real-time RT-PCR assay for routine diagnosis of classical swine fever in wild boar.

The aim of the study was to evaluate a real-time reverse transcription-polymerase chain reaction (RT-PCR) assay for routine diagnosis of classical swine fever (CSF) in wild boar by using 1000 spleen homogenates that were tested previously negative by virus isolation and 26 homogenates from which CSF virus could be isolated. All 26 positive samples in the virus isolation assay also were found to be positive in real-time RT-PCR. Additionally further 10 samples were detected by real-time RT-PCR out of the 1000 negative samples in the virus isolation. With a commercial CSF antigen-ELISA only 14 out of the 36 real-time RT-PCR positive samples could be detected. The sequence analysis of all ten samples that tested positive by real-time RT-PCR and negative by virus isolation revealed CSF virus-specific sequences. Based on the assumption that all samples with a CSF virus-specific sequence or positive in the virus isolation test originated from truly CSF virus-infected wild boar, the following sensitivity values were calculated as antigen-ELISA, 39%; virus isolation, 72% and real-time RT-PCR, 100%. The use of real-time RT-PCR instead of antigen-ELISA and virus isolation as a routine tool for control and eradication of CSF in wild boar populations is recommended.

A universal heterologous internal control system for duplex real-time RT-PCR assays used in a detection system for pestiviruses.

A heterologous in vitro transcript based on a specific primer-probe HEX system was generated as a universal internal control (IC) to improve virus-specific real-time reverse-transcriptase PCR (RT-PCR) assays. By using a set of different primers, several PCR fragments of desired sizes of an in vitro transcript of the enhanced green fluorescent protein (EGFP) gene were generated, and the fragments were detected using a HEX-labelled probe. For long-term storage of the in vitro transcript a special RNA-safe buffer (RSB) was developed. Freezing and thawing of the IC diluted in RSB did not result in any substantial loss of detectable IC copy numbers. The new IC system was used for the first time in a duplex real-time RT-PCR assay for the detection of pestivirus-derived RNA, in particular from bovine viral diarrhea virus (BVDV). Primers and TaqMan probes for the 'panpesti' assay were selected by analysing the consensus sequence of the 5' non-translated region (5' NTR) of more than 600 different pestiviruses. Finally, the optimised primer probe combination showed an analytical sensitivity of less than 10 copies/reaction. In the duplex set-up, the analytical sensitivity of the validated real-time RT-PCR was identical to the sensitivity of the single assay without IC, and the diagnostic sensitivity of the duplex assay was equal or higher if compared to virus isolation.

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.

Long-term studies on maternal immunity for Aujeszky's disease and classical swine fever in wild boar piglets.

The aim of the studies was to fathom the duration and the role of maternal immunity for Aujeszky's disease (AD) and classical swine fever (CSF) in wild boar offspring. In one experiment, two wild boar sows were infected with a low pathogenic pseudorabies virus (PRV) in 1999. A total of 51 offspring was born between 1999 and 2002 and was monitored for PRV maternal antibodies. In a second experiment, the maternal immunity for CSF was analysed. Therefore, a sow was orally vaccinated against CSF using vaccine baits containing the live-attenuated C-strain vaccine. The vaccination took place in January 1999. The sow gave birth to four piglets in 2001 and to two piglets in 2002. With respect to maternal immunity for AD, some piglets reacted positive in the ELISA up to 27-week post-partum while in the neutralization test antibodies were detected up to 15-week post-partum. The calculated half-life of neutralizing antibodies was 21 days. Regarding CSF, the neutralization titres of maternal antibodies dropped continuously reaching values of < or =10 ND50 20-week post-partum. After the 12th week post-partum, most of the sera reacted negative in the ELISA. However, after the third month, low levels of neutralization titres were still detectable. The results are discussed with respect to the epidemiology and control of both diseases in wild boar populations.

Validation of a real-time RT-PCR assay for sensitive and specific detection of classical swine fever.

A fully validated, ready-to-use, real-time reverse transcription-polymerase chain reaction (RT-PCR) assay, multiplexed for simultaneous detection of an internal control, for the simple and rapid diagnosis of classical swine fever (CSF) was developed. Primers and FAM-labeled TaqMan-probes specific for classical swine fever virus (CSFV) were selected from the consensus sequence of the 5' non-translated region (5' NTR) of 78 different CSFV strains. For determining analytical sensitivity, an in vitro transcript (T7-PC3alf) of the 5' NTR was constructed and tested. In addition, the T7-PC3alf transcript was further used as a positive control and a standard for quantitation of CSFV genome copies. A second heterologous in vitro transcript based on a specific primer-probe HEX-system was designed as an internal positive control for the RNA isolation step and RT-PCR. By using limited primer concentrations for the internal control, no adverse effects on the sensitivity of the CSF-system could be observed, and the newly designed duplex real-time RT-PCR proved to have a sensitivity of approximately eight copies. The primer-probe combination selected was strictly CSFV-specific and no amplification was observed in all non-CSFV pestiviruses tested.

Classical swine fever (hog cholera) in wild boar in Europe.

Classical swine fever (CSF) is of increasing concern in Europe where wild boar appear to play an important epidemiological role. In most parts of the continent, demographic trends are on the increase, due to improvement in game management. As a result of higher densities, populations become more susceptible to various infectious diseases, among which CSF is cause for particular concern. Wild boar do not appear to be a classic reservoir in most cases, but nevertheless may perpetuate foci of infection over the long term, constituting a real threat for the pig farming industry. Since the infection does not appear to spread easily in natural populations of free-ranging wild boars, control of the disease may be feasible. However, most of the appropriate measures, such as banning hunting, are not considered acceptable. Consequently, the expertise of wildlife disease specialists is required to help solve the problem when it occurs.

Are low-density granulocytes the major target cells of classical swine fever virus in the peripheral blood?

The target cells of classical swine fever (CSF) virus in the peripheral blood of pigs infected with recent field isolates from Germany were studied. Eight weaned pigs were inoculated oronasally with the CSF virus field isolate Visbek/Han 95 and three weaners were inoculated with the isolate Losten/Freese 98. All pigs showed severe clinical signs typical of CSF and died or had to be euthanized between 9 and 24 days post-infection (dpi). The first cells in the peripheral blood which became infected with CSF virus were mixed granulocytes (a combination of low- and high-density granulocytes). These cells yielded the highest infectivity for PK 15 cell cultures. On day 7 post-infection, the peripheral blood mononuclear cell (PBMC) fraction was virus positive, while the peripheral blood leucocyte (PBL), peripheral blood T lymphocyte (PBT) and high-density granulocyte fractions were either negative or their infectivity was lower than the infectivity of the PBMC fraction. These results indicate that PBMC contain more virus-positive cells than other fractions of leucocytes. These findings may also have diagnostic implications for the detection of CSF virus in blood samples. Because PBMC showed the highest infectivity in the early stages of CSF, it should be the sample of choice for CSF virus isolation.

Classical swine fever (CSF) marker vaccine. Trial II. Challenge study in pregnant sows.

The efficacy of two marker vaccines against classical swine fever (CSF) was tested in a large scale laboratory trial in several National Swine Fever Laboratories (NSFL) of the EU member states. The vaccines were: BAYOVAC CSF Marker (Vaccine A) from Bayer, Leverkusen, Germany and PORCILIS PESTI (Vaccine B) from Intervet, Boxmeer, The Netherlands. At the NSFL of Belgium, The Netherlands and Germany experiments were carried out to examine the ability of the vaccines to prevent transplacental transmission of CSF virus. In Belgium and The Netherlands pregnant sows were vaccinated once and challenged with virulent CSF virus 14 days later, which was around day 60 of gestation. At the NSFL in Germany sows were vaccinated twice, on days 25 and 46 of pregnancy and were challenged fourteen days after booster vaccination (day 60 of gestation). Apart from minor inflammatory reactions in some sows, no reactions post vaccination were noticed in either vaccine group. Sows vaccinated with Vaccine A were better protected against clinical CSF than sows vaccinated with Vaccine B. The antibody response after vaccination with Vaccine A was more pronounced than after vaccination with Vaccine B. After single vaccination six out of eight sows vaccinated with Vaccine A and all eight sows vaccinated with Vaccine B had viraemic piglets. After double vaccination one out of four litters from sows vaccinated with Vaccine A and four out of five litters from sows vaccinated with Vaccine B were found to be viraemic. However, both vaccines reduced the transmission probability significantly (Vaccine A: P=0.004, Vaccine B: P=0.024) after booster vaccination. However, Vaccine A appeared in this regard more potent as the estimated probability of fetal infections was lower. Nevertheless the risk of virus spreading after vaccination via transplacental transmission is still present and has to be addressed from an epidemiological point of view.

Detection of classical swine fever virus in semen of infected boars.

During the Classical Swine Fever (CSF) epidemic in 1997 in the EU member states Germany, Italy, Spain and The Netherlands, boars in an artificial insemination (AI) centre were found to be infected with CSF virus. This raised a question of epidemiological importance which could not be answered immediately. Can CSF virus be shed by semen of infected boars and what conclusions concerning the risk of spreading CSF infection by semen can be drawn. Experimental studies were conducted to answer this question. Four young boars were infected with a CSF field virus isolate from Germany, which had been characterised in a previous animal experiment. Semen was collected at least every other day after infection. The semen was subjected to the standard diagnostic procedure for the detection of CSF virus and to semen quality assessment. The boars were euthanized at day 8, 12, 16 and 21 post infection, respectively. A post mortem examination was done and organ samples were taken from the CSF reference organs and genital organs for the detection of virus and antigen. The course of CSF infection of the boars was mild but detectable during the second week of infection. CSF virus could be isolated from semen of two animals during the pyrexic phase and from the epididymis but not from the testes. Since CSF virus shedding via semen could be proven, it was concluded that the disease may also be transmitted by artificial insemination. However analysis of semen in cell culture for the presence of CSF virus is not suitable as a routine method for CSF diagnosis.

Transient classical swine fever virus infection in wild boar piglets partially protected by maternal antibodies.

An experimental study was conducted to investigate the clinical course of classical swine fever (CSF) in wild boar piglets partially protected by maternal antibodies. Five healthy wild boar piglets with a low serum titre of colostral antibodies against CSF virus were challenged with virulent CSF virus at the age of three months. Apart of reduced food intake and diarrhoea no major clinical symptoms were noticed after challenge. These signs were seen during the second and third week of infection, afterwards the piglets recovered completely. CSF virus could be re-isolated from blood samples taken on day 12 and day 19 post challenge. From blood samples taken later on and from the organ material taken at post mortem examinations no CSF virus could be isolated anymore. It can be concluded that the presence of maternal antibodies influences the clinical course of CSF in terms that the outcome is rather transient than lethal. Such wild boar could play a crucial role in the spread of CSF virus and might contribute to the maintenance of long lasting epizootics.