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.

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.

BVD virus isolation techniques for routine use in cattle herds with or without previous BVD history.

Buffy coats of 1074 cattle were tested for BVD virus using the usual longterm-cultivation (LTC) in bovine kidney monolayer cell cultures (7 days) whereby 268 BVD virus carriers could be detected. Serum samples collected simultaneously from the same animals were examined by means of a shortterm-cultivation (STC) procedure of only two days in stationary macroplate cell cultures. Using this method only 172 amongst the former 268 BVD virus carriers were found. Of the remaining 96 serum samples from animals positive in buffy coats leucocytes by LTC and negative in sera by STC, further 19 cattle were found to be viraemic when the sera were additionally tested by LTC. These results are discussed with regard to the antibody level and the age of the animals. The reduced sensitivity of STC of sera is considered in relation to the favourable time and cost factor. STC of serum samples in connection with the serological results on a herd basis proved to be valuable for the examination of cattle of more than 6 months of age but not for calves below 6 months. This was particularly true in cattle herds with no previous BVD history.

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.

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.

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.

Effects of classical swine fever virus infection on the porcine leukocyte subsets.

The effects of classical swine fever (CSF) virus infection on the porcine leukocyte subsets were investigated by flow cytometry in acute, chronic and convalescent forms of the disease. The virus antigen could be first detected in the monocytes on postinfection (p.i.) day 10 while in the lymphocytes on p.i. day 13. It could be established that the ratio of CD6+ cells decreased until p.i. day 6, but afterwards it started to increase and reached different values. The CD4+CD8+, the CD8+ and the CD6- cells were obviously higher virus positive than the CD4+ and the CD4-CD8-subsets, but essentially all subsets could be infected. The ratio of CD8+ cells increased during the disease, while the number of double positive cells decreased, and that of the CD4+ cells was variable. The viral antigen could be detected in a lower percentage of the CD4+CD8+, CD8+, CD6+ and CD6- cells of the pigs affected with the chronic form of the disease than in those with the acute form. During the experiments no viral antigen could be detected in the leukocytes of the pig that became convalescent, though the changes in its leukocyte subsets were very similar to those seen in pigs in which the viral antigen could be detected. The studies have revealed that essentially all leukocyte subsets can be infected with the CSF virus, but in very different amounts.

Evaluation of a gp 55 (E2) recombinant-based ELISA for the detection of antibodies induced by classical swine fever virus.

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against classical swine fever virus (CSFV) based on the competition of the serum antibodies with a CSFV-specific monoclonal antibody directed against the viral glycoprotein gp 55 (E2), has been evaluated. A total of 553 sera obtained from pigs experimentally infected in groups with different pestiviruses were included in this study. The ELISA was applied to a group of sera collected from pigs prior to pestivirus inoculation and therefore expected to have no detectable CSFV neutralizing antibodies. The specificity of the ELISA was calculated to range between 93% and 98%. The sensitivity of the ELISA was determined by testing predominantly sera of pigs exposed to CSFV and exhibiting CSFV neutralizing antibodies. When the cut-off level was reduced by 10%, the average sensitivity increased from 60% to 70% for the groups of sera tested. The ELISA was able to discriminate between CSFV and bovine viral diarrhoea virus (BVDV) induced antibodies.

[Transplacental BVD virus transmission after experimental inoculation of goats in different pregnancy stages]. / Transplazentare BVD-Virusübertragung nach experimenteller Inokulation von Ziegen in unterschiedlichen Trächtigkeitsstadien.

Eight groups of altogether 25 goats without neutralizing antibodies against BVD virus, were inoculated either intranasally or intranasally and subcutaneously with two different BVD virus isolates during different stages of gestation. In all 18 goats inoculated within the first 78 days of gestation an abortion and foetal death rate of approximately 100% occurred. Only one goat gave birth to a clinically healthy kid. The other seven goats which were inoculated after the 78th day of gestation showed also a high foetal death rate. Only two of them gave birth to clinically healthy kids. Neutralizing antibodies against BVD virus could be detected in blood samples drawn from 14 kids born at normal term including stillborn and non-viable offsprings. BVD virus was reisolated from different organs taken from seven foetuses. It was not possible to isolate BVD virus from any of the normal offsprings.

[Bovine virus diarrhea (BVD)--diagnostic determination of cattle with persistent BVD virus infection]. / Bovine Virusdiarrhoe (BVD)--Diagnostische Ermittlung von Rindern mit persistenter BVD-Virusinfektion.

Buffy coats of 1074 cattle were examined for regard to BVD virus using the usual long-termed-cultivation (LTC) in bovine kidney monolayer cell cultures (7 days) whereby 268 carriers could be detected. Serum samples collected simultaneously from the same cattle were examined by means of a short-termed-cultivation (STC) of only two days in stationary macroplate cell cultures. Using this method only 172 carriers were found. Amongst 96 serum samples from animals positive in LTC of buffy coat leucocytes and negative in the STC further 19 cattle were found to be viraemic when the sera were tested by LTC, too. Thus 77 of a total of 268 serum samples (28.7%) taken from viraemic cattle turned out to be negative in the STC as well as the LTC. These results are discussed with regard to the antibody level and the age of the animals. The reduced sensitivity is considered in relation to the favourable time and cost factor. STC of serum samples in connection with the serological results proved to be valuable for the examination of cattle of more than 6 months of age but not for calves below 6 months.

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.

Classical swine fever in wild boar (Sus scrofa)--experimental infections and viral persistence.

A pregnant wild boar and two wild boar weaners were inoculated intranasally with a field isolate of classical swine fever virus (CSFV) recently derived from a diseased domestic pig. The clinical, pathological and haematological findings noted in the young wild boars were comparable to those in domestic weaner pigs inoculated with the same virus isolate. Both wild boars showed the acute haemorrhagic form of CSF, one animal died 18 days post inoculation (p. i.) and the second one had to be euthanized when moribund two days later. The wild boar sow did not show any signs of illness p. i. but seroconversion was noticed. Twenty-eight days p. i. birth was given to six clinically healthy offsprings. One of the newborn proved to be viraemic until death when 39 days of age. Except for poor growth no other symptoms were noticed in this piglet. The non-viraemic litter mates remained healthy, although they had close contact to the persistently infected piglet. High titres of neutralizing antibodies against CSFV were measured in the serum samples of these offsprings. All findings were more or less in accordance with observations previously made in domestic pigs when infected with CSFV around 85 to 90 days of gestation. The wild boar was calculated to have been inoculated at about 87 to 92 days of gestation.

[Comparison of different BVD virus strains for their use in the differential diagnosis of classical swine fever--an attempt to standardize neutralization tests]. / Vergleich verschiedener BVDV-Stämme für Zwecke der Differentialdiagnose in der KSP-Serologie--ein Beitrag zur Standardisierung des Neutralisationstestes.

Six bovine virus diarrhoea (BVD) virus strains were tested in the neutralization test for their use in the differential diagnosis in classical swine fever (CSF) serology. The aim of the investigation was to find a suitable BVD virus strain guaranteeing a safe differentiation of CSF- and BVD virus induced antibodies using permanent cell cultures (PK-15, MDBK). For test purposes the neutralizing antibody titres of 73 defined test sera were titrated against the CSF virus strain Alfort/187 as well as the BVD virus strains Grub, Paplitz, NADL, 1138/69, Stendal, 10421/Han 94). Tests were repeated fivefold. The level of mean antibody titres, the differences in titre to the homologous pestivirus strain, the standard deviation and the variation coefficient served as test criteria. The BVD virus strains Grub and NADL yielded the best results. In view of harmonization and standardization the BVD virus strain NADL in connection with a standard protocol for neutralization tests is recommended for the differential diagnosis in CSF serology. Due to the adaptation of the permanent cell-lines PK-15 and MDBK to horse serum a further source of contamination with non cytopathogenic BVD viruses under routine conditions can be excluded.

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.

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.

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.

Does porcine reproductive and respiratory syndrome virus potentiate classical swine fever virus infection in weaner pigs?

Fifteen 6-week-old crossbred weaners weighing about 12 kg each were randomly divided into three groups of five animals each. One group of pigs was inoculated first with porcine reproductive and respiratory syndrome (PRRS) virus and then 3 days later with CSF virus. The second group received classical swine fever (CSF) virus, while the third group was inoculated with PRRS virus only. The aim of the experiment was to determine whether a primary PRRS virus infection influences the clinical outcome of experimentally induced CSF in young pigs. The PRRS virus infected weaners developed mild respiratory symptoms and recovered completely. All five weaners which were inoculated with CSF virus only showed severe clinical signs typical of the acute form of CSF. One pig had to be killed 15 days post-inoculation (p.i.); the remaining four died between the 18th and 22nd day p.i. The clinical course of the animals inoculated with both viruses was slightly different from that of the pigs that received only CSF virus. Four out of five pigs from the PRRS/CSF group became febrile and viraemic earlier than the animals which received CSF virus only. These pigs had to be killed 15-17 days post CSF virus inoculation. One animal in this group survived the acute phase of CSF and recovered completely. It was concluded that the observed divergences of the clinical courses would not have been noticed under field conditions. Therefore these findings cast doubt on the relevance of PRRS virus infection potentiating significantly the clinical outcome of CSF in young pigs.