Classical swine fever (CSF) marker vaccine

Veterinary Microbiology 83 (2001) 85–106 Classical swine fever (CSF) marker vaccine Trial I. Challenge studies in weaner pigs Åse Uttenthala,*, Marie-Frédérique Le Potierb, Luis Romeroc, Gian Mario De Miad, Gundula Floegel-Niesmanne a Danish Veterinary Institute for Virus Research, Lindholm, DK-4771 Kalvehave, Denmark b Agence Française de Sécurité Sanitaire des Aliments, Ploufragan, France c Centro de Investigación en Sanidad Animal, 28130 Valdeolmos, Madrid, Spain d Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche, Via G Salvemini 1, 06100 Perugia, Italy e Community Reference Laboratory for Classical Swine Fever, Institute of Virology, Hannover Veterinary School, Bünteweg 17, 30559 Hannover, Germany Received 18 April 2000; received in revised form 9 March 2001; accepted 9 April 2001 Abstract Two commercial marker vaccines against classical swine fever virus (CSFV) and companion diagnostic tests were examined in 160 conventional pigs. To test the vaccines in a ‘‘worst case scenario’’, group of 10 weaners were vaccinated using a single dose of an E2 (gp55) based vaccine at days
21,
14,
10 or
7, and subsequently challenged at day 0. The challenge virus was CSFV 277, originating from a recent outbreak of classical swine fever (CSF) in Germany. In all groups, only 5 out of 10 pigs were challenged; the remaining 5 pigs served as vaccinated contact controls. Also, three control groups, each consisting of 10 non-vaccinated pigs, were challenged in parallel to the vaccinated animals. CSFV could be isolated from all non-vaccinated pigs. Among these pigs 40% displayed a chronic course of the infection (virus positive for more than 10 days). Pigs vaccinated 21 or 14 days before challenge displayed no clinical signs of CSFV after challenge. However, they were still able to replicate CSFV when challenged, as measured by reisolation of CSFV from leukocytes of the directly challenged pigs. CSFV could be isolated from the leucocytes of 25% of the pigs vaccinated 21 days before challenge and 50% of the pigs vaccinated 14 days before challenge. Chronic infection was not observed, but transmission to one vaccinated contact pig occurred. From all pigs vaccinated 10 or 7 days before challenge, CSFV could be reisolated. We observed a chronic course of infection in 5% of pigs vaccinated 10 days before challenge and in 30% of pigs vaccinated 7 days before challenge. The mortality rate was 20% in the pigs vaccinated 10 days before challenge, and varied between 20 and 80% in pigs vaccinated 7 days prior to challenge. The contact animals had lower mortality (0–20%) than directly challenged pigs, probably mirroring the * Corresponding author. Tel.: þ45-55-86-03-93; fax: þ45-55-86-03-00. E-mail address: aau@vetvirus.dk (Å. Uttenthal). 0378-1135/01/$ – see front matter # 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 1 3 5 ( 0 1 ) 0 0 4 0 9 - 6 86 Å. Uttenthal et al. / Veterinary Microbiology 83 (2001) 85–106 delayed time point of infection. There was thus some protection against clinical illness by both marker vaccines, but not a solid protection against infection and virus shedding. The efficacy of the vaccine was best if used 3 weeks before challenge and a clear correlation between time interval from vaccination to challenge and the level of virus shedding was observed. Each vaccine had its own accompanying discriminatory ELISA, but 18% of the virus positive pigs never seroconverted in these tests. # 2001 Elsevier Science B.V. All rights reserved. Keywords: Classical swine fever; Marker vaccine; Subunit vaccine; Hog cholera; Challenge study; Discriminatory ELISA 1. Introduction The Scientific Veterinary Committee (SVC, 1997) recommended in 1997 that CSF marker vaccines could be an additional tool of disease control especially in pig dense areas but further research was considered necessary. Consequently the large scale marker vaccine trial was financed by the EU Commission (98/S 192-129475/EN) and organised by the EU Reference Laboratory for CSF (CRL). The objective of the trial was to test the two available marker vaccines mimicking emergency field conditions and to evaluate their corresponding discriminatory tests. The results of the trial are published in three parts. This study concerns the use of marker vaccines in weaner. The use of marker vaccines were likewise studied in sows (Depner et al., 2001); finally the reliability of the discriminatory ELISA was analysed (FloegelNiesmann, 2001). Classical swine fever virus (CSFV) is a pestivirus within the family Flaviviridae (Moennig, 1990). CSFV is the cause of a highly contagious disease (classical swine fever (CSF)) occurring in domestic pigs in several European countries (Williams and Matthews, 1988; Koenen et al., 1996; Widjojoatmodjo et al., 1999; Stadejek et al., 1997). The recent outbreaks of CSF have all been controlled by stamping out, and have caused major economic losses to the pig industry and the European community (Elbers et al., 1999; Sharpe et al., 2001). The present legislation does not allow prophylactic vaccination within the EU, but if an outbreak has been established, animals kept near to the diseased herds may be emergency vaccinated according to the council directive 80/217/EEC. The traditional CSFV vaccines are live attenuated vaccines based on the lapinised CSFV strain C. If prophylactic vaccination was used, it would drastically interfere with the serological surveillance performed in all EU member states, because of an inability to differentiate vaccinated from infected animals. To avoid this, several groups have examined the efficacy of subunit marker vaccines based on single CSFV proteins expressed from virus vectors (van Zijl et al., 1991) or in the baculovirus expression system (van Rijn et al., 1996, 1999). The pestivirus particle contains four structural proteins, namely, the nucleocapsid protein (C) and three glycoproteins, Erns (gp44/48), E1 (gp33) and E2 (gp55) (Thiel et al., 1991; Bruschke et al., 1997). The most immunogenic proteins in CSFV are the E2 and the Erns, the latter having been shown to exhibit RNase activity (Hulst et al., 1998). Pigs vaccinated with either E2 or Erns resisted a lethal CSFV challenge, whereas animals Å. Uttenthal et al. / Veterinary Microbiology 83 (2001) 85–106 87 vaccinated by E1 did not (van Rijn et al., 1996; Konig et al., 1995). Both vaccines contained baculovirus expressed E2 as antigen. In the accompanying ELISA systems, antibodies to Erns are detected using recombinant Erns as antigen. Thus, such ELISA are not expected to detect vaccinated pigs, but should detect animals that have undergone CSFV infection, also if prophylactically vaccinated. It would be ideal if vaccines could be used for emergency vaccination in the face of a CSFV outbreak. In that case, a high probability would exist of animals being exposed to CSFV shortly after vaccination. To mirror this in the experimental set-up, weaner pigs were vaccinated shortly before experimental challenge. One single vaccination dose was administered either 21, 14, 10 or 7 days prior to challenge of half of the animals, the remaining pigs serving as vaccinated in-contact-controls. In order to increase the number of animals in the test, and to eliminate confounding factors associated with a given laboratory, each experiment was done independently in two laboratories. Thus, the study described in this paper was done at four National Swine Fever Laboratories (NSFL) and the EU Reference Laboratory for CSF. The aim of the study was to investigate if marker vaccine given under extreme conditions would change the clinical course of disease and reduce virus multiplication from vaccinated and challenged weaners. Also, we wished to examine the ability of the accompanying ELISA kits to differentiate vaccinated from infected animal. 2. Materials and methods 2.1. Experimental set-up Four laboratories performed four independent vaccination-challenge studies. For each laboratory, two different vaccines were used. The ability of each vaccine to reduce spread of CSFV was tested by injecting one single vaccine dose for each pig at days
21,
14,
10 or
7, followed by challenge at day 0. Each vaccine was tested in the same manner at two independent laboratories; in addition, two laboratories included challenge studies of an unvaccinated control group of pigs and a solitary control group was also challenged at the Community Reference Laboratory in Hannover, Germany. The studies of vaccination 7 and 10 days before challenge were performed in Perugia, Italy and Valdeolmos, Spain, whereas the studies on vaccination 14 and 21 days before challenge were performed in Ploufragan, France and Lindholm, Denmark. The challenge studies of non-vaccinated control groups were performed in France and Denmark. In each country, pigs of local landrace aged 5–6 weeks were purchased from a local herd of good health status. The 190 pigs were divided into 19 groups of 10 each, and numbered and blood sampled to assure lack of antibodies to CSF, bovine viral diarrhoea (BVD) and porcine reproductive and respiratory syndrome virus (PRRSV). All pigs were seronegative to all the above mentioned viruses apart from the French group, where maternally derived antibodies to BVD were present in about 50% of the pigs. The anti-BVD titres were decreasing, and at the time of challenge only 20% of the pigs were still seropositive, showing very low neutralising titres to BVD (titre values of 10–30). To assure that the pigs were not infected by BVD virus, all French pigs were tested for pestivirus before challenge, using a closed one-tube RT-PCR (McGoldrick et al., 1999). Briefly RNA was extracted 88 Å. Uttenthal et al. / Veterinary Microbiology 83 (2001) 85–106 from 140 ul serum obtained before challenge using a commercial Quiagen kit; 50 ul of RNA was analysed in the RT-PCR. At days
21,
14,
10 and
7, 10 pigs were vaccinated intramuscularly using the BAYOVAC1 CSF marker or PORCILIS1 PESTI vaccine (Table 1). Before the challenge at day 0, five pigs from each group were transferred to another pen or unit, and challenged by oral or intranasal administration of CSFV Paderborn (see below). Twenty-four hours later, the pigs were returned to their vaccine groups. Thus, five pigs in each vaccinated group were challenged intranasally (Spain, France, Italy) or orally (Denmark) or oronasally (Germany), whereas the remaining five pigs served as vaccinated in-contact pigs. The titres of the challenge material used by the laboratories varied between 102.54 and 105 (Table 1). In France, Germany and Denmark a control group of non-vaccinated pigs was included. The pigs in this control group were treated in parallel to the vaccinated pigs. Thus, half of the animals were given oral, oronasal or nasal challenge. Blood samples were obtained in plain tubes and in EDTA-K tubes approximately every second day for 2 weeks, then weekly until day 42 (day 37 for the Italian pigs). All these blood samples were analysed for neutralising antibodies, Erns antibodies and live virus. Rectal temperatures and clinical signs were recorded daily. Fever was defined as temperatures above 408C. Moribund pigs were euthanised; the remaining pigs were killed by captive bolt stunning and exsanguination 5–6 weeks after challenge. 2.2. Vaccines Two subunit marker vaccines were tested in parallel. Both vaccines were based on the CSFV envelope glycoprotein E2 produced in the baculovirus expression system, and adjuvanted in oil emulsion. The PORCILIS1 PESTI January 1999 (Intervet, The Netherlands) contained 120 ELISA units of E2 antigen per dose. The BAYOVAC1 CSF marker E2/98/B001 (Bayer, Germany) contained 32 mg of E2 glycoprotein per dose. Both vaccines were injected intramuscularly in the neck, using 2 ml per dose as recommended by the manufacturers. Both manufacturers recommended for prophylactic use a vaccination of 6 weeks old piglets followed by a booster injection 4 weeks later. 2.3. Virus strains The challenge material was supplied by the Reference Laboratory in Hannover as ampoules with freeze-dried third PK-15 cell passage of a German CSF field strain, isolated in 1997 from the Paderborn area (CSF 277, genotype 2.1) (Greiser-Wilke et al., 1998). Further passages in PK-15 cells were done in some laboratories (Table 1). For neutralisation tests, the Alfort 187 CSFV strain (Dahle and Liess, 1995) was used. 2.4. Virus isolation The methods used were adapted from the council directive 80/217/EEC Annex A and the OIE Manual for Standards of Diagnostics and Vaccines. Only a short description is detailed here. Table 1 Comparison of animal experiment protocols for the participating laboratoriesa Number of pigs Further passages of the challenge virusb Challenge route and CSFV dose per pigc Number of passages used for reisolation Reisolation of CSFV from blood done on France Ctrl and pcd
14 and
21 50 3  PK-15 Intranasally 102.54 TCID50 2 Plasma or heparin stabilised full-bloodd Denmark Ctrl and pcd
14 and
21 50 No further passage Orally 104.4 TCID50 1 Leukocytes isolated using dextran sulphate Spain pcd
7 and
10 40 1  PK-15 Intranasally 105 TCID50 3 Leukocytes isolated using NH4Cl Italy pcd
7 and
10 40 1  PK-15 Intranasally 105 TCID50 2 Buffy coat Germany Ctrl 10 1  PK-15 Oronasally 103 TCID50 2 Leukocyte isolated using dextran sulphate a pcd: post-challenge day; pcd
14 and
21: vaccination 14 and 21 days before challenge; pcd
7 and
10: vaccination 7 and 10 days before challenge; Ctrl: no vaccination before challenge. b The challenge material was supplied by the Community Reference Laboratory in Hannover as freeze-dried third PK-15 cell passage of a German CSFV field strain isolated in Paderborn in 1997, and further passaged as shown. c The advised challenge dose was 103 TCID50, the actual challenge dose per pig were determined by back-titration of the challenge material. d Plasma (from EDTA stabilised blood) was used for all French virus isolation attempts from vaccinated pigs pcd 0–42. The non-vaccinated control pigs were analysed using plasma pcd 0–12 and days 35 and 42. Non-vaccinated control pigs were analysed at days 14, 17 and 21 using Heparin stabilised full-blood. Å. Uttenthal et al. / Veterinary Microbiology 83 (2001) 85–106 Place of trial, and vaccination-challenge interval 89 90 Å. Uttenthal et al. / Veterinary Microbiology 83 (2001) 85–106 Isolation of CSFV was done from stabilised blood samples using buffy coat or leukocytes isolated by the dextran sulphate or NH4Cl method. Washed leukocytes were freeze/thawed before virus isolation was attempted. In the French pigs isolation was initially performed on plasma from EDTA stabilised blood. However, as the detection of virus in the vaccinated groups turned out to be very low, a further sample of heparin stabilised blood was obtained from the non-vaccinated control pigs day 14, 17 and 21 after challenge and virus isolation was performed on this full-blood sample for the control group only. All CSFV isolations on vaccinated French pigs were done on plasma from EDTA stabilised blood. One to three blind passages were assayed in PK-15 cells as shown in Table 1. The fixed cultures were stained using polyclonal pig serum or monoclonal antibodies to reveal the presence of CSFV. 2.5. Test for neutralising antibodies Serum samples were tested for antibodies against CSF Alfort 187 using a direct neutralising peroxidase- or fluorescence-linked antibody assay in PK-15 cells. For all laboratories, an inhibiting dilution of test serum of 1:10 was scored positive (Hyera et al., 1987; Anonymous, 1997). 2.6. Discriminatory ELISA CEDITEST-ELISA (Institute for Animal Science and Health (ID-DLO), Lelystad, The Netherlands) was performed according to the manufacturers instructions on serum obtained from pigs vaccinated with the BAYOVAC1 CSF. CHEKIT CSF marker Erns ELISA (Dr. Bommeli Ag, Hoechst Roussel Vet Diagnostics, Liebefeld-Bern) was performed according to the manufacturers instructions on serum from pigs vaccinated with the PORCILIS1 PESTI. Both tests were blocking ELISA testing for antibodies to Erns. A separate paper assesses the performance of these ELISA kits (Floegel-Niesmann, 2001). 2.7. Statistical calculations The Student’s t-test was used to examine the significance of differences in body temperatures between vaccinated and non-vaccinated groups at days 14, 21 and 28. As only two laboratories had both vaccinated and non-vaccinated pigs this analysis was only possible for the Danish and French groups (vaccination 21 and 14 days before challenge). For the comparison all pigs in the vaccine group were analysed, regardless of their status as contact pigs or infected pigs. For the experiments in Spain and Italy (vaccination 10 and 7 days before challenge) unfortunately no unvaccinated groups were included, and comparison to other non-vaccinated groups, such as the German group, would not be statistically reliable. Therefore, no statistics were performed on the temperature data of the Spanish, Italian and German groups. The relative risk of a contact animal contracting CSF infection from a challenged animal in the same group was calculated. For the estimation of the relative risk the numbers of CSF infected contact pigs (Table 7) and the number of uninfected contact pigs in each group Å. Uttenthal et al. / Veterinary Microbiology 83 (2001) 85–106 91 were added to the other relevant factors (vaccine challenge interval, vaccine brand). A contact animal was regarded as CSF infected, if the Erns ELISA gave a positive result at least once, or CSFV could be isolated from the blood of the animal at least once. Influence of the vaccine brand (Intervet and Bayer) was first calculated by adding all animals from all groups. Further both vaccine brands were compared by adding groups 7 and 10 in Spain and Italy. The same calculation was done comparing groups 14 and 21 in Denmark and France. Influence of the vaccination-challenge interval was calculated by comparing group 10 with 7 taking both vaccines together. Also group 21 was compared with group 14 adding both vaccine brands together. A calculation for comparing group 7 with 14 or group 10 with 21 is not appropriate because the two groups were processed in two different laboratories and different effects could not be estimated separately. Risk calculations were performed using the SAS-procedure PROC FREQ. This procedure utilises approximative confidence bounds for the relative risk. For additional interpretation the level attained (P-value) using Fischer’s exact test was calculated (SAS Institute Inc., 1989; Kreienbrock and Schach, 1997). 3. Results Differences in procedures performed at the laboratories are shown in Table 1. The inoculum used for challenge was centrally distributed as a freeze-dried material, to ensure the maximal reproducibility in this study. Further cell culture passages were optional. The advised challenge dose was 103 TCID50 per pig, but back-titration revealed differences in the challenge dose used. The French pigs were given the lowest CSF dose, 102.54. The German pigs were given an intermediate dose of 103 TCID50 per pig, whereas the Danish pigs were challenged using 104.4 TCID50 CSFV per pig. The challenge doses in Spain and in Italy were the highest used (105 TCID50 per pig). 3.1. Non-vaccinated control group Four to fourteen days after challenge, marked clinical signs such as fever, apathy and inappetence were observed among the non-vaccinated pigs (Table 2). Four weeks after challenge, some of the pigs showed central nervous signs and intracutaneous bleeding. However, these clinical signs, considered to be indicative of CSF, were not observed in all of the non-vaccinated pigs. The only consistent clinical sign was fever. Mortality was slightly lower in the contact group than in the directly challenged group. Thus, in France two directly challenged and one contact pig died. All German directly challenged pigs died, whereas only three of five contact pigs died. In the Danish experiment 3 directly challenged and two control pigs died; the death of pigs is shown on Fig. 1. A chronic course of infection (defined as a pig being virus positive for more than 10 days) was observed in 7 of 15 directly challenged and 5 of 15 contact pigs. The mean rectal temperatures from days 0 to 42 after challenge are given in Fig. 1. The directly challenged pigs had fever from post-challenge day (pcd) 4–7, whereas 92 Å. Uttenthal et al. / Veterinary Microbiology 83 (2001) 85–106 Table 2 Detection of antibodies and virus in non-vaccinated groups of five directly CSFV challenged and five contact pigsa Place of trial Francec Germany Denmark First occurrence of neutralising antibodies (pcd) Onset of viraemia (pcd) Number of animals exhibiting a chronical course of CSFVb Challenged Contact Challenged Contact Challenged Contact 10–14 18–22 21–>42 17–21 15–32 28–>42 4–6 4–6 6–21 10–14 15 6–21 2 4 1 1 4 0 a pcd: post-challenge day; CSFV could be isolated from leukocytes in all pigs. Chronic course of infection: pigs being virus positive for more than 10 consecutive days. c Non-vaccinated pigs were analysed using plasma (from EDTA stabilised blood) pcd 0–12 and days 35 and 42. Heparin stabilised full-blood was analysed at days 14, 17 and 21. b the contact animals did not develop fever until day 10–14. In both the French and the Danish experiment the non-vaccinated groups had significantly higher mean body temperature than vaccinated groups at pcd 14 (P < 0:02, Fig. 1). In the Danish experiment this significant difference was present until pcd 28, whereas in the French experiment pigs had significantly higher temperatures (P < 0:05) pcd 21 when compared to vaccinated groups, except for the Intervet vaccinated 21 days before challenge which was just below P < 0:05. In the French experiments no significant difference was observed on day 28. The German experiment consisted of non-vaccinated pigs only; they had fever from day 7. From all challenged and contact pigs, virus could be reisolated from leukocytes. The initial date of seroconversion, and whether the CSF displayed a chronic course, was comparable in challenged and contact animals (Table 2). 3.2. Weaners vaccinated 21 days before challenge Pigs vaccinated 21 days prior to challenge did not develop significant fever following challenge (P > 0:05) compared to pigs vaccinated 14 days before challenge (Table 3, Fig. 1). No clinical signs were observed in pigs vaccinated with Bayer or Intervet vaccines. As both vaccines contained the E2 protein, most pigs had neutralising antibodies (Rümenapf et al., 1991) at pcd 0, probably preventing CSFV isolation from plasma. Leukocytes, however, were washed prior to release of virus by lysis. The impact of neutralising antibodies will thus be decreased. For the Danish experiment the non-vaccinated control group was housed in a separate pen in the same isolation room as the vaccinated pigs, and all pigs were challenged the same day. Horizontal transmission of CSFV through air might have occurred (Dewulf et al., 2000) thus increasing the infection pressure on the vaccinated contact pigs. In the French experiment, no virus could be reisolated from plasma, whereas in the Danish pigs, virus could be isolated from the leucocytes of two challenged, Bayer vaccinated pigs and three challenged, Intervet vaccinated pigs. One Danish Bayer vaccinated pig died 33 days after challenge. A chronic course of infection was not Å. Uttenthal et al. / Veterinary Microbiology 83 (2001) 85–106 93 detected in any pigs in this group. No virus could be reisolated from any vaccinated contact pigs. All serum samples were analysed by the discriminatory Erns ELISA. In the challenged, Bayer vaccinated groups, 7 out of 10 pigs had transient positive reactions in the Fig. 1. Fever in groups of pigs following CSF challenge. The daily mean temperature of each group of 10 pigs is given. Open symbols are used for Intervet vaccinated pigs, filled symbols for Bayer vaccinated pigs. Pigs vaccinated 21 days before challenge (&); pigs vaccinated 14 days before challenge (~); pigs vaccinated 10 days before challenge (*); pigs vaccinated 7 days before challenge (^); non-vaccinated pigs (). ( ) Pigs dying from CSF during the study. For France and Germany, mortality only occurred in non-vaccinated animals. For Denmark a single vaccinated pig died at pcd 33 (not shown in the figure), and five non-vaccinated pigs died as shown in the figure. For Italy and Spain the symbols for dead pigs are explained in the figure. 94 Å. Uttenthal et al. / Veterinary Microbiology 83 (2001) 85–106 Fig. 1. (Continued ). accompanying Erns ELISA. The contact pigs vaccinated with the Bayer vaccine generally remained Erns ELISA negative. Only 1 out of 10 pigs became Erns ELISA positive shortly before the end of the experiment. Challenged pigs vaccinated with the Intervet vaccine all turned Erns ELISA positive between pcd 10 and 21. Already on pcd 2 all Danish pigs scored Erns ELISA positive, probably due to unspecific reactions. The French Intervet contact pigs remained Erns ELISA negative, whereas the Danish Intervet contact pigs all turned Erns ELISA positive from pcd 14 to 35. 95 Å. Uttenthal et al. / Veterinary Microbiology 83 (2001) 85–106 Fig. 1. (Continued ). 3.3. Weaners vaccinated 14 days before challenge No clinical signs or significant fever was observed in these animals following challenge (Table 4, Fig. 1). Neutralising antibodies to CSF were detectable in 40% of the pigs at the time of challenge. At day 6 (at the time point when virus could be isolated from Table 3 Virus isolation and detection of antibodies in groups of five pigs directly challenged 21 days after vaccination and five vaccinated contact pigsa Place of trial vaccine France Bayer France Intervet Denmark Bayerd Denmark Intervetd CSFV isolated from leukocytes #pos samples/#pos pigs (onset of virus detection, pcd) First occurrence of neutralising antibodies (pcd)b No. of pigs Erns positive First occurrence of Erns antibodies (pcd) Challenged Contact Challenged Contact Challenged/ contact Challenged Contact 0c 0c 2/2 (8–28) 6/3 (6) 0c 0c 0 0 0–2 0–6 0–6 6–28 3/0 5/0 4/1 5/5 10–>42 10–12 12–42 10–21e >42 >42 42–>42 14–35e 0–2 0–4 0 0–21 a pcd: post-challenge day; >42: no antibodies were detected during the 42 days observation period; Erns positive: detection of antibodies in the accompanying discriminatory ELISA. b For the first pig and the last pig in the group. c Virus isolation was performed on plasma obtained from EDTA stabilised blood. d Vaccinated and control pigs were housed in the same isolation unit. Thus, we cannot exclude that virus transmission might have occurred between groups. e Unspecific reactions to Erns in all pigs pcd 2. 96 Å. Uttenthal et al. / Veterinary Microbiology 83 (2001) 85–106 Table 4 Virus isolation and detection of antibodies in groups of five pigs directly challenged 14 days after vaccination and five vaccinated contact pigsa Place of trial vaccine France Bayer France Intervet Denmark Bayerd Denmark Intervetd CSFV isolated from leukocytes #pos samples/#pos pigs (onset of virus detection, pcd) No. of pigs First occurrence of neutralising antibodies Erns positive (pcd)b First occurrence of Erns antibodies (pcd) Challenged Contact Challenged Contact Challenged/ contact Challenged Contact 0c 3/2c (4–6) 2/2 (6) 9/5 (6–10) 0c 0c 0 1/1 (28) 0–6 4–8 0–6 8–10 0–4 6–17 0 8–42 5/2 5/5 4/2 5/5 12–42 12–17 12–>42 12–14e 35–>42 17–28 35–>42 21–28e a pcd: post-challenge day; >42: no antibodies were detected during the 42 days observation period; Erns positive: detection of antibodies in the accompanying discriminatory ELISA. b For the first pig and the last pig in the group. c Virus isolation was performed on plasma obtained from EDTA stabilised blood. d Vaccinated and control pigs were housed in the same isolation unit. Thus, we cannot exclude that virus transmission might have occurred between groups. e Unspecific reactions to Erns in all pigs pcd 2. non-vaccinated pigs) all Bayer vaccinated pig had neutralising antibodies, whereas only 4 of 20 Intervet vaccinated pigs had neutralising antibodies to CSFV. From challenged pigs vaccinated with the Bayer vaccine, virus could not be reisolated from pigs in the French group, whereas two of the Danish challenged pigs were viraemic. No virus could be reisolated from any of the Bayer vaccinated contact pigs. Intervet vaccinated pigs had viraemia in 7 of 10 challenged pigs, and in one contact pig. No pigs displayed a chronical infection. The Erns ELISA detected antibodies in 9 out of 10 Bayer vaccinated challenged pigs. The single pig that did not at any time have a positive reaction in the Erns ELISA was viraemic at pcd 7. In the contact pigs, 4 of 10 became Erns ELISA positive. However, for all pigs the positive reaction was of an intermittent nature. For the Intervet vaccinated pigs, all 20 pigs became Erns ELISA positive. The Erns ELISA reaction in the Intervet vaccinated pigs was of a more consistent nature than observed in the Erns ELISA test of Bayer vaccinated pigs. 3.4. Weaners vaccinated 10 days before challenge Pigs vaccinated 10 days before challenge showed clinical signs sporadically, 17 of 20 challenged pigs had brief periods of fever (Table 5, Fig. 1), often coinciding with viraemia. In the contact animals only 12 of 19 pigs had fever, and no virus was isolated from the leukocytes of the seven pigs without fever. As they were vaccinated shortly before challenge, the neutralising antibody reaction was only observed around pcd 7 in the challenged group. Different laboratories observed different responses in the contact 97 Å. Uttenthal et al. / Veterinary Microbiology 83 (2001) 85–106 Table 5 Virus isolation and detection of antibodies in groups of five pigs directly challenged 10 days after vaccination and five vaccinated contact pigsa Place of trial vaccine Italy Bayer Italy Intervet Spain Bayerc Spain Intervet CSFV isolated from leukocytes #pos samples/#pos pigs (onset of virus detection, pcd) First occurrence of neutralising antibodies (pcd)b No. of pigs First occurrence of Erns positive Erns antibodies (pcd) Challenged Contact Challenged Challenged/ contact Challenged Contact 22/5 17/5 19/5 22/5 2/1 (18) 0/0 3/2 (8–10) 16/5 (6–14) <7 <7–17 6–10 8–14 5/5 5/4 3/2 5/4 11–25 12–23 10–>42 8–14 18–36 23–>37 42–>42 21–28 (9–11) (4–9) (2–4) (4–6) Contact <7 12–17 8–28 14–21 a pcd: post-challenge day; >42: no antibodies were detected during the 42 days observation period; Erns positive: detection of antibodies in the accompanying discriminatory ELISA. b For the first pig and the last pig in the group. c Only four contact pigs. animals: all the Italian, Bayer vaccinated contact pigs had neutralising antibodies from day 7 (the first blood sample analysed), whereas the Spanish Bayer vaccinated contact pigs seroconverted later (pcd 8–28). Reisolation of CSFV was possible from the leukocytes of all 10 Bayer vaccinated challenged pigs. In the contact pigs of the Bayer vaccinated group, only three of nine pigs were viraemic, and only brief fever was observed. Generally, the viraemic period was prolonged, compared to the pigs vaccinated 21 or 14 days before challenge, lasting about a week in the individual pig, reflected in the larger number of positive findings. In the Italian group, one challenged and one contact pig died, both had been viraemic shortly before death. Eight of 10 Bayer vaccinated, challenged pigs became Erns ELISA positive. The positive reactions occurred late (the majority of pigs at days 21–35), and often the Erns ELISA reverted to negative in serum samples obtained a few days later from the same pig. The two pigs staying Erns ELISA negative were viraemic for more than a week. Seven out of nine Bayer vaccinated contact pigs became Erns ELISA positive, from days 18 to 42 postchallenge. Viraemia was not detected in the two Erns ELISA negative contact pigs. The Intervet vaccinated, challenged pigs all had viraemia and fever. One Spanish pig died at pcd 18 and one Italian pig died following a chronic course pcd 35. Neutralising antibodies appeared pcd 7–21. There was no correlation between initial detection of virus and occurrence of neutralising antibodies. In the contact pigs, CSFV was not reisolated from the Italian pigs, whereas viraemia was present in all the Spanish pigs. One contact pig from Spain died pcd 18, coinciding with the appearance of neutralising antibodies. For the surviving contact pigs, neutralising antibodies were detected pcd 12–21 in both countries. All 10 challenged Intervet vaccinated pigs became Erns ELISA positive from days 8 to 23 after challenge, 8 out of 10 Intervet vaccinated contact pigs became Erns ELISA positive shortly after, from days 21 to 37 post-challenge. 98 Å. Uttenthal et al. / Veterinary Microbiology 83 (2001) 85–106 Table 6 Virus isolation and detection of antibodies in groups of five pigs directly challenged 7 days after vaccination and five vaccinated contact pigsa Place of trial vaccine Italy Bayer Italy Intervet Spain Bayerd Spain Intervet CSFV isolated from leukocytes #pos samples/#pos pigs (onset of virus detection, pcd) First occurrence of neutralising antibodies (pcd)b No. of pigs First occurrence of Erns positive Erns antibodies (pcd) Challenged Contact Challenged Contact Challenged/ contact Challenged Contact 15/5 22/5 27/5 25/5 0 0 14/5 (4–12) 16/5 (6–12) 7 9–>27c 8–>26 10–42 7 12–17 10–21 14–28 5/0 3/2 2/5 4/3 11–16 17–27 35–>42 4–>14 >37 31–>37 21–42 12–>42 (9–11) (4–7) (4) (4) a pcd: post-challenge day; >42: no antibodies were detected during the 42 days observation period; Erns positive: detection of antibodies in the accompanying discriminatory ELISA. b For the first pig and the last pig in the group. c Two pigs of five remained seronegative. d Only four contact pigs. 3.5. Weaners vaccinated 7 days before challenge Pigs in this vaccine group displayed the highest number of cases of chronical infection, as four Intervet vaccinated, challenged pigs and two Bayer vaccinated, challenged pigs were virus positive for more than 10 days (Table 6). Only one contact pig (Spain, Invervet vaccinated) was virus positive for more than 10 days. All Spanish pigs in the Bayer vaccinated group had fever, whereas only two Italian, Bayer vaccinated pigs, both challenged, had fever; 4 of 10 Bayer vaccinated challenged pigs died (days 15, 17, 24 or 27) due to diarrhoea and dehydration caused by CSFV infection. All Bayer vaccinated contact animals survived, but some of them showed mild clinical signs of CSF. Virus could be isolated from all Bayer vaccinated challenged pigs. A single Italian Bayer vaccinated pig died pcd 15, without showing specific clinical signs of CSF. All Spanish Bayer vaccinated contact animals were viraemic, and surprisingly showed a more acute course than did the Bayer vaccinated challenged pigs. In the Italian group, however, no virus could be isolated from the contact pigs. Neutralising antibodies to CSF were detected already on day 7 after challenge in the Italian Bayer vaccinated group, whereas the response in the Spanish Bayer vaccinated group was severely depressed: only two challenged pigs mounted neutralising antibodies. In Spain all surviving Bayer vaccinated pigs were Erns ELISA positive. In Italy, only the challenged pigs became Erns ELISA positive, and all Italian Bayer vaccinated contact animals remained Erns ELISA negative. In Spain as well as in Italy, only Bayer vaccinated animals from the leukocytes of which CSFV could be isolated were Erns ELISA positive. In Spain, all Intervet vaccinated pigs had fever and became viraemic, with longer periods of viraemia in the challenged than in the contact pigs (Table 6). The Italian Intervet vaccinated, challenged pigs had fever and viraemia, and no virus could be 99 Å. Uttenthal et al. / Veterinary Microbiology 83 (2001) 85–106 Table 7 Overview on the CSF status of contact pigsa Place of trial vaccine Spain Italy Denmark France Vaccine challenge interval (days) Intervet vaccine 7 10 5/5 2/4 5/5 4/5 Bayer vaccine 14 5/5 5/5 21 7 10 5/5 0/5 2/4 4/5 5/5 0/5 14 21 2/5 2/5 2/5 0/5 a Pigs from the leukocytes of which CSFV was isolated or which had at least one positive reaction in the Erns ELISA is regarded as CSF infected. The numerator gives the number of CSFV infected pigs and the denominator gives the number of all pigs in each group. isolated from the Italian Intervet vaccinated, contact pigs. Clinical signs of CSF were predominant, with neurological signs, depression and diarrhoea. In the challenged Intervet vaccinated group, 7 of 10 pigs died (pcd 7, 14, 14, 22, 23, 24 and 33) whereas only one of nine contact animals died. Neutralising antibodies were detected in challenged pigs from 9 to 17 dpc in the Italian pigs, whereas the antibody production in the Spanish pigs was delayed up till 42 dpc. The Intervet vaccinated, contact animals all mounted a neutralising antibody response, starting from days 12 to 28. Seroconversion in the Erns ELISA was very fast but inconsistent in the Spanish pigs, already day 4 after challenge Erns ELISA positive pigs were detected. Five of seven challenged, Intervet vaccinated pigs became Erns ELISA positive days 12–17. Only five out of eight surviving contact animals became Erns ELISA positive. 3.6. Statistical evaluation of reduction of spread by each vaccine The relative risk for contact animals to contract CSFV infection was calculated from the values shown in Table 7, the definition of infection is described in Section 2. The relative risk of CSFV infection of a contact animal 10 days post-marker vaccination is not significantly reduced (1.25) compared to 7 days post-marker vaccination of the herd (95% confidence intervals 0.83–1.89; P ¼ 0:476). The relative risk of CSFV infection of a contact animal 21 days post-marker vaccination is not significantly reduced (0.50) compared to 14 days post-marker vaccination (95% confidence intervals 0.26–0.97; P ¼ 0:056). Comparison of vaccine brands in all groups in all countries revealed that the relative risk of CSFV infection for a contact animal after vaccination in the herd with Intervet vaccine is significantly higher (1.82) compared to the risk of CSFV infection after vaccination with the Bayer vaccine (95% confidence intervals 1.23–2.70; P ¼ 0:002). Comparison of vaccine brands in groups 7 and 10 revealed that the relative risk of CSFV infection for a contact animal after vaccination of the herd with Intervet vaccine is not significantly higher (1.46) compared to the risk of CSFV infection after vaccination with the Bayer vaccine (95% confidence intervals 0.95–2.25; P ¼ 0:151). 100 Å. Uttenthal et al. / Veterinary Microbiology 83 (2001) 85–106 Comparison of vaccine brand together in groups 14 and 21 revealed that the relative risk of CSFV infection for a contact animal after vaccination with Intervet vaccine is significantly higher (2.50) compared to the risk of CSFV infection after vaccination with the Bayer vaccine (95% confidence intervals 1.22–5.11; P ¼ 0:01). 4. Discussion With regard to OIE list A diseases, such as CSF, a general non-vaccination policy has been followed in the EU countries, using stamping out in case of disease introduction into any of the member states. In recent years, however, stamping out CSFV outbreaks has caused serious economical consequences in some member states. Furthermore, a strict stamping out policy without vaccination can in fact prolong the duration of nation wide CSFV epidemics, because once herds display serological or clinical signs of CSFV infection, CSFV shedding and spread to neighbouring naive herds may already have taken place for several weeks. Therefore, two marker vaccines that might be available on the market in the near future were analysed for their ability to stop the transmission of CSFV among pig herds. CSFV challenge in non-vaccinated weaners was performed in three laboratories. In all groups, a high morbidity and mortality was observed in challenged animals, and CSFV transmission to contact animals was detected (Table 2). These findings validated the experimental set-up with regard to the efficacy assessment of marker CSFV vaccines to reduce morbidity, mortality and viral spread during CSFV epidemics. When comparing the efficacy of the two E2 marker vaccines, no major differences were found, both vaccines significantly reduced the spread of CSFV to native controls (Table 7). Although the statistical evaluation is in favour of the Bayer vaccine, the calculated figures should not be over interpreted. The discriminatory ELISA accompanying the Bayer vaccine had severe deficiencies in sensitivity (Floegel-Niesmann, 2001) and consequently some Erns antibody positive animals might have not been detected, thus giving the Bayer vaccine a better statistical result. The shift towards a subclinical course of CSF infection and the reduction of viraemia, which were evident with both vaccines, are of major concern for the purpose of disease eradication. Therefore, the discussion will mainly focus on the time interval between vaccination and challenge on the protective effect of the vaccine. The group of pigs vaccinated 21 days before challenge served as a ‘positive control’ for the efficacy of vaccination. In fact, the group vaccinated 21 days before challenge (Table 3) exhibited reduced percentages of animals from the plasma and leukocytes of which CSFV could be isolated post-challenge, compared to non-vaccinated animals (Table 2). In the French pigs, no virus could be isolated from the directly challenged pigs, and still 8 of 10 pigs had Erns antibodies. The reason is probably that antibodies present after vaccination in the plasma neutralises the virus, thus the presence of virus is underestimated. Still vaccination markedly reduced replication of the challenge virus, and this prevented mortality in directly challenged animals, as well as reduced levels of viral transmission to vaccinated contact animals (Tables 3 and 7). It is always a theoretical concern with vaccination, whether the protective effect that curtails acute virus infection may instead cause increased Å. Uttenthal et al. / Veterinary Microbiology 83 (2001) 85–106 101 number of animals that become chronically infected; such animals could act as long-term virus carriers. To investigate this, we examined the number of unvaccinated animals which displayed a protracted course of CSFV infection, defined as the number of animals from the leukocytes of which CSFV could be isolated for 10 days or more (Table 2). Vaccination reduced the occurrence of chronically infected pigs corresponding to the time interval between vaccination and challenge; thus all pigs vaccinated 21 or 14 days before challenge displayed an acute infection. Among pigs vaccinated 10 days before challenge only one pig had a chronic course whereas 6 out of 20 directly challenged and 1/19 contact animals were virus positive for more than 10 days. Vaccination, therefore, did not increase the number of chronic cases. Furthermore, there was a reverse correlation between the interval between vaccination and challenge, and the contact animals to the pigs vaccinated 7 days before challenge had a reduced number of chronic courses, possibly due to the delayed infection time in the contact pigs. Having established the level of protection induced by the E2 marker vaccines under what we consider to be optimal conditions, we investigated their efficacy when used closer to the challenge time, a situation which is likely to occur in the field if such vaccines are to be used for emergency vaccination in the face of CSFVoutbreaks. When used at 14 days prior to challenge (Table 4), vaccination still afforded significant reductions in the number of animals from the blood and leukocytes of which CSFV could be isolated. Again, this likely reflected a vaccine-induced curtailment of viral replication in directly challenged animals, resulting in reduced mortality figures as well as reduced levels of CSFV transmission to vaccinated contact animals. However, if vaccination was done 10 days prior to challenge (Table 5), viral replication levels in directly challenged animals were similar to those observed in non-vaccinated animals following CSFV challenge, and this resulted in significant mortality in the vaccinated, directly challenged group. Also, the level of CSFV shedding from infected animals was indistinguishable from that observed in the nonvaccinated, challenged group (Table 3). The situation outlined above for vaccination 10 days before challenge was even more pronounced if vaccination was done 7 days before challenge. A number of important conclusions arise from these results: (i) when used 21 days prior to challenge, vaccination afforded reduction in morbidity and mortality following subsequent challenge with a virulent field CSFV isolate. However, our results also indicated that even when administered as early as 21 days before challenge, protection was not complete, with five directly challenged, Danish vaccinated pigs having CSFV positive leukocytes. Compared to former studies of the protective effect of E2 these results may appear less convincing. A number of factors could explain the difference in protection by E2 vaccines observed in ours and previous studies. For example, earlier studies have focused on prophylactic vaccination, using a vaccination-challenge interval of 4 weeks (Bouma et al., 1999; Rümenapf et al., 1991) or vaccinated twice (van Rijn et al., 1996; van Zijl et al., 1991), whereas we challenged the animals 7–21 days after a single vaccination. Also the above mentioned challenge studies were done using a lethal dose of the highly virulent Alfort or Brescia CSFV strains. In this study, a recent field isolate was used for challenge. The presence of virus evidenced in isolated leukocytes obtained from a larger volume of blood, a method that is more sensitive than analysing oral swabs (Moormann et al., 2000). (ii) When used 21 days prior 102 Å. Uttenthal et al. / Veterinary Microbiology 83 (2001) 85–106 to challenge, vaccination markedly reduced viral shedding from directly challenged to contact animals. The ability of vaccination to prevent viral shedding is of crucial importance during emergency vaccination campaigns, where the aim of vaccination is not to protect individual animals, but to restrict viral spread in the population as a whole. (iii) The present study allowed us to define the shortest vaccination-challenge interval where vaccination may still be expected to provide reduction in viral shedding from directly challenged animals. This period was approximately 14 days for both the two commercially available E2 marker vaccines used in the present study. If vaccine was administered shorter than 14 days prior to challenge, a high mortality was found and no reduction in viral shedding from directly challenged animals was seen. However, considering that the marker vaccines are comparable to killed vaccines as opposed to the live C-strain vaccine, the reduction of virus shedding and mortality 14 dpi is remarkable. Although the different laboratories have standardised diagnostic procedures, evaluated at an annual interlaboratory comparison test, a number of experimental variables differed between labs, as shown in Table 1 and discussed below. In all countries, local landrace breeds were used. The breed of the pigs might influence the course of a CSFV infection (Depner et al., 1997), explaining the differences in the severity of clinical signs observed between the different labs participating in this study. Also, differences in the inoculation procedure (nasal, oral), and the number of cell culture passages of the challenge CSFV stock and the management of the pigs might have influenced the course of infection. Additionally, the challenge dose would be expected to influence disease severity. Interestingly, the challenge dose did not correlate to disease severity in the challenged, nonvaccinated pigs (Table 2). The German pigs experienced an acute infection with high mortality (80%) within the 42-day observation period. Both the French and Danish pigs, displayed a milder, but more chronic, infection with a lower mortality and a later onset of clinical signs. Interestingly the clinical signs in the French pigs were mainly central nervous with ataxia, whereas the Danish pigs exhibited skin haemorrhages, apathy and diarrhoea. In both countries, only some of the pigs showed clinical signs, whereas others had only a few days of CSFV isolation in leukocytes and then seemingly recovered from disease. Finally, before challenge, the French pigs had low neutralising BVD titers. The BVD seropositive pigs were equally distributed in control and vaccine groups, as well as within group distributions to challenge or contact pigs in order not to bias the study. Also, at the time of challenge, the BVD titer was low and no neutralising antibodies to CSFV were present in any of the French pigs at the time of vaccination. Furthermore pigs with maternal BVD antibodies were also present in the non-vaccinated control group; no reduction or delay in virus isolation was observed compared to the Danish of German pigs. Therefore, the interference of maternal BVD immunity is disregarded. By using plasma for virus isolation in all vaccinated French pigs some virus positive pigs must have passed unnoticed. The limited finding of two virus positive pigs in the 20 French pigs vaccinated 14 days before challenge, was followed by appearance of Erns antibodies in 17 pigs (Table 4). Such findings stresses that the virus isolation results from these pigs are underestimated, as expected. Another difference, which might influence the Danish experiment, was that all pigs were kept in the same room. That is, the virus excreted from the non-vaccinated control pigs Å. Uttenthal et al. / Veterinary Microbiology 83 (2001) 85–106 103 could have infected the vaccinated contact pigs as shown recently (Dewulf et al., 2000). This might give an overestimation of the number of contact pigs infected. However, looking at the spread of CSFV in pigs vaccinated 14 days before infection does not reveal differences (Table 7). We therefore, decided to evaluate both the French and the Danish pigs in this trial in spite of these different procedures. The lack of a non-vaccinated control group in the Spanish and Italian experiments impair the statistical evaluation of the pigs infected 10 or 7 days after vaccination. The observed differences between the severity of disease at these two laboratories therefore, cannot be properly addressed. A crucial problem in CSFV diagnostics is the immunosuppressive effect of the disease (Knoetig et al., 1999). Even when the pigs were vaccinated before challenge with CSFV, a proper immune response was not mounted. The impact of disease on the pigs vaccinated 7 or 10 days before challenge might explain the difference in the results in Spain and Italy. The Spanish pigs were severely immunosuppressed, and the neutralising antibodies in challenged pigs were impaired. Excretion of CSFV starts 4–7 days after challenge and seemingly this limited delay improves the prognosis for the contact animal, as shown by the decrease in mortality rate in-contact animals compared to challenged penmates. Interestingly this effect was most pronounced in pigs vaccinated 7 days prior to challenge. Furthermore several pigs were Erns ELISA negative even though CSFV had been detected in the leukocytes (Table 8). This finding appeared not to be restricted to any of the vaccines, as detailed in Table 8. The proportion of pigs without an Erns antibody was higher in the groups vaccinated 7 or 10 days before challenge. The lack of Erns ELISA reactivity in virus positive animals could be due to virus multiplication rapidly causing the death of the pig, as opposed to ‘‘true’’ immunosupression in live animals. To analyse this, the time span (days) from detection of virus in the leukocytes until death or seroconversion in the Erns ELISA has been detailed (Table 8). The animals which did produce Erns antibodies mostly did so 0–4 days after virus was detected in leukocytes, whereas CSFV-induced mortality mostly occurred from 5 days after CSFV was detected in leukocytes and onwards (Table 8). This showed that many of the animals, which did not develop Erns antibodies, must in fact have had time enough to do so before they died. Thus, our data indicated that some animals failed to mount Erns responses prior to death, likely due to CSFV-induced immunosuppression. The presence of such individuals is of great practical importance, because they are not detected by the existing Erns ELISA systems (Table 8). This, combined with the lack of clinical signs in the pigs, may cause ongoing CSFV epidemics to go unnoticed. Our data allow an estimation of the frequency of such animals: 11 of 60 (18%) pigs did not seroconvert in the Erns ELISA despite surviving for at least 14 days after CSFV was detected in their leukocytes. Measuring the neutralising antibodies are of little value in this study, as neutralising antibodies appear as a response to vaccination. Antibodies are observed approximately 21 days after vaccination. If pigs are challenged earlier than 14 days after vaccination the immunosuppressive effect of CSFV seem to interfere with the production of neutralising antibodies. This study examined the feasibility of marker vaccination to reduce spread of CSFV during an epidemic. Marker vaccines are only useful with a companion diagnostic test, which discriminate vaccinated from infected animals. However, in our hands, the discriminatory Erns ELISA systems produced variable results, for example, some animals, from the leukocytes of which CSFV could be isolated did not score an Erns positive 104 Table 8 Correlation between detection of CSFV in the leukocytes and detection of Erns antibodies (abs) after CSFV challenge of vaccinated pigs or CSFV spread to contact pigs Serum: Erns ELISA Bayer 21 Positive 7 1 Intervet 21 Bayer 14 Intervet 14 Bayerb 10 Intervet 10 Negative Positive Negative Positive Negative Positive Negative Positive Negative Positive Negative Positive Negative Positive Negative 11 12 5 12 6 12 0 4 2 4 1 0 5 2 2 1 3 0 1 1 8 0 11 2 14 1 12 3 10 5 Bayer Intervet a b 7 b 7 Unspecific reactions to Erns in all pigs pcd 2. Only 19 pigs were in the group. No. of pigs with CSFV negative leukocytes CSFV positive leukocytes Time from CSFV detection to Erns antibodies (abs) or death of the pig Erns abs 2 days later Died 5 days after CSFV detection Erns abs 4, 4, 6 days latera Erns abs 5 days later Died 36 days after CSFV detection Erns abs 0, 2, 6, 6, 6, 6, 10, 11 days latera Erns abs 0, 0, 2, 2, 3, 6, 14, 31, 32, 33, 34 days later Died 38, 38 days after CSFV detection Erns abs 2, 5, 6, 8, 8, 8, 10, 11, 13, 13, 14, 19, 20, 20 days later Died 10 days after CSFV detection Erns abs 0, 2, 2, 2, 7, 9, 9, 31, 30, 31, 30, 38 days later Died 13, 20, 23 days after CSFV detection Erns abs 0, 0, 2, 8, 8, 9, 13, 13, 13, 16 days later Died 3, 5, 10, 17, 36 days after CSFV detection Å. Uttenthal et al. / Veterinary Microbiology 83 (2001) 85–106 Vaccination-challenge interval (days) Vaccine used Å. Uttenthal et al. / Veterinary Microbiology 83 (2001) 85–106 105 antibody result, although they recovered from infection. Antibodies to Erns were not consistently produced in all CSFV infected animals for successive samples taken within a few weeks from the same animal (data not shown, the performance of the Erns ELISA will be detailed in a separate manuscript, Floegel-Niesmann, 2001). Our conclusion is, that the use of marker vaccines cannot be recommended, mainly due to shortcomings of the discriminatory tests available at the time the experiment was performed (January–June 1999). On the other hand, the effect in reducing the virus multiplication and thus the infection pressure by vaccination in the herd is evident. Thus, the solid basis is founded, but the vaccines and accompanying serological tests have to be refined further before they can be considered an efficient tool in emergency field vaccination, for example, by using live, attenuated marker vaccines, as recently described (Widjojoatmodjo et al., 2000). Acknowledgements We thank Intervet, The Netherlands and Bayer, Germany for supplying us with the vaccines. The Institute for Animal Science and Health (ID-DLO), The Netherlands and Hoechst Roussel Vet Diagnostics, Liebefeld-Bern, Germany donated the kits for the discriminatory ELISA. The project was funded by the Commission of the European Community. We thank Marion Petersen, Carina Malm, Jonna Jensen, Bent Eriksen, Heidi Clausen, Roland Cariolet, Evelyne Hutet, Anne-France Saintilan, Sébastien Le Gal, Claudia Pellegrini, Angela Saavedra, Mercedes Moyano, Belen Vazquez, Gabi Mueller, Sebastian Fischer and Guenther Thiem for excellent technical assistance and animal handling. We thank Dr. Beyerbach and Prof. Kreienbrock from the Institute of Biometry and Epidemiology, School of Veterinary Medicine Hannover, for the statistical calculations. Dr. Martin Oleksiewicz is thanked for critical revision of the manuscript. References Anonymous, 1997. 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