Kinetics and type of immune response following intranasal and subcutaneous immunisation of calves

Protection of animals against respiratory infections has long been known to depend on respiratory mucosal immunity. However, few studies have been reported on the immune response following intranasal (i.n.) immunisation with non-living, soluble antigens. This study determined the kinetics of the humoral and cellular immune responses in calves after i.n. immunisation with Limulus haemocyanin (LH) with cholera toxin adjuvant, or subcutaneous (s.c.) immunisation with LH in incomplete Freund's adjuvant. A proliferative response of peripheral blood mononuclear cells cultured in vitro with LH was observed in animals immunised 7-10 days after i.n. and s.c. immunisations with no significant differences between the two immunised groups. LH -specific antibody was present in the serum of animals immunised s.c. (IgM, IgG1 and IgG2) and i.n. (IgA). Although significant IgA responses were observed, i.n. immunisations in cattle with soluble protein antigens and cholera toxin as an adjuvant did not induce a strong systemic immune response.     

Antibody isotype profiles in serum and circulating antibody-secreting cells following mucosal and peripheral immunisations of sheep

The isotype-specific antibody responses of sheep immunised with keyhole limpet hemocyanin by a peripheral route (intramuscular (i.m.) injection) were compared to those induced by immunisation via different mucosal routes: (1) intra-nasal spray; (2) rectal deposition with cholera toxin; (3) injection into the mucosa of the small intestine or rectum. Antigen-specific IgG1 antibodies were induced in the i.m., intra-intestinal and intra-rectal injection groups and in a proportion of the cholera toxin immunised sheep, but not in the intra-nasal immunisation group. IgA was the only antibody isotype detected in serum collected from the intra-nasal immunisation group. No significant differences in serum IgA levels were detected in any of the mucosal immunisation groups as compared to the i.m. injection group. In contrast, analysis of the in vitro antibody profiles secreted by circulating antibody-secreting cells (ASC) revealed significantly higher IgA responses in the supernatants from all mucosal immunisation groups. This suggests that the measurement of antibodies secreted by circulating ASCs may be a better correlate of local mucosal responses in ruminants, as has been previously demonstrated in human studies. In addition to IgG1 and IgA responses, immunisation by direct injection of antigen formulations into the intestinal and rectal mucosa were the only groups to induce consistently high IgG2 antibodies in serum and ASC cultures.     

Potential of DNA-mediated vaccination for equine herpesvirus 1.

The potential of DNA-mediated immunisation to protect against equine herpesvirus 1 (EHV-1) disease was assessed in a murine model of EHV-1 respiratory infection. Intramuscular injection with DNA encoding the EHV-1 envelope glycoprotein D (gD) in a mammalian expression vector induced a specific antibody response detectable by two weeks and maintained through 23 weeks post injection. Immune responses were proportional to the dose of DNA and a second injection markedly enhanced the antibody response. EHV-1 gD DNA-injected mice developed neutralising antibodies, and a predominance of IgG2a antibodies after the DNA injection was consistent with the generation of a type 1 helper T-cell (Th1) response. Following intranasal challenge with EHV-1, mice immunised with 50 microg of EHV-1 gD DNA were able to clear virus more rapidly from lung tissue and showed reduced lung pathology in comparison with control mice. The data indicate that DNA-mediated immunisation may be a useful strategy for vaccination against EHV-1.     

Effect of transcutaneous immunization with co-administered antigen and cholera toxin on systemic and mucosal antibody responses in sheep

Direct application of antigens to skin together with an adjuvant, a procedure called transcutaneous immunization (TCI), can induce systemic immune responses in mice, humans, cats and dogs. In previous studies we found that cholera toxin (CT) applied topically on unbroken skin induces systemic antibody and lymphocyte proliferative responses in sheep. The current study examined whether concurrent administration of CT and tetanus toxoid (TT) delivered transcutaneously could induce specific antibody responses to both antigens in sheep. Antibodies to both TT and CT were induced by TCI although antibody titres in serum to TT were higher in sheep receiving TT plus alum by intramuscular injection (n=5) than TT plus CT by TCI (n=5). The ratio of IgG1/IgG2 antibody to TT in serum was near unity, and the route of immunization, TCI versus injection, did not influence this ratio. In contrast, the ratio of IgG1/IgG2 antibody differed significantly between the two antigens, TT and CT, delivered by TCI, with a higher proportion of IgG1 antibody in serum to CT than TT. Antibody to TT was detected in lung washes from TCI and injection groups, with IgG1 predominating over IgG2 in both groups. IgA antibodies to CT and TT were detected in sera of CT and TT-immunized groups respectively but in lung washes IgA antibody to TT was detected only in the injection group. Results show that TCI induced systemic antibody responses to CT and the co-administered antigen TT, whereas no evidence was obtained for mucosal IgA responses following TCI.     

Antibody response in bovine pharyngeal fluid following foot-and-mouth disease vaccination and, or, exposure to live virus

Samples of pharyngeal fluid and serum were collected from cattle after exposure to live foot-and-mouth disease (FMD) virus (with or without prior vaccination) or after subcutaneous vaccination with inactivated virus. The pharyngeal fluid samples were examined for FMD neutralising activity and specific anti-FMD IgG, IgM and IgA antibodies. The neutralising activity of the serum was also monitored. A peak of neutralising activity which occurred in the pharyngeal fluid of unvaccinated cattle seven days after virus exposure corresponded to a rise in specific IgM and IgA antibodies. This peak appeared to be due to serum and tissue fluid escaping from the damaged mucosa during the acute inflammatory phase of infection. At later stages (20 to 60 days after virus exposure) the pharyngeal fluid neutralising activity corresponded to a rise in specific IgA antibodies, suggesting that active local antibody production was taking place. The pharyngeal fluid neutralising activity detected after revaccination with oil emulsion or aqueous vaccines, without exposure to live virus, corresponded to a rise in specific IgG and IgM antibody levels and this may have been due to serum transudation.     

Antigenicity of structural components from porcine transmissible gastroenteritis virus

Pregnant sows were inoculated with inactivated transmissible gastroenteritis virus and with preparations of virus surface projections and subviral particles derived by detergent treatment of the virus. Neutralising antibody was demonstrated in serum and colostrum from animals that received whole virus or preparations of surface projections whereas subviral particles failed to stimulate neutralising antibody formation. Similar results were obtained with serum from rabbits inoculated with whole virus and structural components. All three preparations stimulated the formation of agglutinating antibodies, as demonstrated by sedimentation analysis and filtration studies with radiolabelled virus.The immunoglobulin classes responsible for neutralising antibody activity in sows inoculated by the intramammary route were examined. In each case where the immunoglobulin class was determined, IgG was found. One sow that received surface projections also had IgA with neutralising activity in her colostrum. In contrast, infection of sows with live whole virus resulted in neutralising antibody of the IgG, IgM and IgA classes.     

A new inactivated BVDV genotype I and II vaccine. An immunisation and challenge study with BVDV genotype I

An inactivated vaccine containing BVDV I and II strains (PT810; BVDV I, and 890; BVDV II) and using different adjuvants and antigen dosages was tested in a cattle challenge model. Groups of six healthy, seronegative cattle were vaccinated twice with a low dose (10(6.6) TCID(50) PT810 and 10(7.2) TCID(50) 890) vaccine with the adjuvant Bay R1005 or a high dose (10(7.8) TCID(50) PT810 and 10(8. 2) TCID(50) 890) vaccine with two different adjuvants (Bay R1005 or Polygen). Thirty-eight days after the second vaccination, immunised animals (n=18) and non-vaccinated control animals (n=3) were challenged intranasally with 10(6) TCID(50) BVDV strain PT810. For a period of 16 days, virus was isolated from blood leukocytes and nasal swabs, and neutralising antibody titres were determined.The induction of antibodies following immunisation was strongly dependent on the antigen dosage in the vaccine. The high dose formulation induced high serum neutralising antibody titres against both genotypes of up to 32000 after the second immunisation. Animals with neutralising antibody titres >512 (n=14) did not show any marked leukopenia after challenge and only very little or no virus could be isolated from blood leukocytes and/or nasal swabs when compared to control cattle. Furthermore, some of these animals did not show any boost of neutralising or even NS3-specific antibodies, which renders viral replication unlikely and thus would prevent infection of the fetus. Both adjuvants (Bay R1005 or Polygen) were similarly efficient and induced nearly identical antibody responses. In contrast, four of the six low dosage vaccinates had a marked leukopenia and viraemia as well as detectable nasal virus shedding for several days.We conclude that the selected strains and the system of vaccine preparation with high BVDV antigen dosages and highly efficient new adjuvants provide an effective means of protection against BVDV I infections. Investigations to demonstrate the protection against BVDV II infections, the duration of immunity and the ability of fetal protection by using the high dose vaccine in a fetal challenge model will follow.     

Duration of protective immunity and antibody responses in cattle immunised against alcelaphine herpesvirus-1-induced malignant catarrhal fever

ABSTRACT: Protection of cattle from alcelaphine herpesvirus-1 (AlHV-1)-induced malignant catarrhal fever (MCF) has been described previously, using an attenuated virus vaccine in an unlicensed adjuvant. The vaccine was hypothesised to induce a protective barrier of virus-neutralising antibody in the oro-nasal region, supported by the observation of high titre neutralising antibodies in nasal secretions of protected animals. Here we describe further analysis of this vaccine strategy, studying the effectiveness of the vaccine formulated with a licensed adjuvant; the duration of immunity induced; and the virus-specific antibody responses in plasma and nasal secretions. The results presented here show that the attenuated AlHV-1 vaccine in a licensed adjuvant protected cattle from fatal intranasal challenge with pathogenic AlHV-1 at three or six months. In addition, animals protected from MCF had significantly higher initial anti-viral antibody titres than animals that succumbed to disease; and these antibody titres remained relatively stable after challenge, while titres in vaccinated animals with MCF increased significantly prior to the onset of clinical disease. These data support the view that a mucosal barrier of neutralising antibody blocks infection of vaccinated animals and suggests that the magnitude of the initial response may correlate with long-term protection. Interestingly, the high titre virus-neutralising antibody responses seen in animals that succumbed to MCF after vaccination were not protective.     

Maturation of functional antibody affinity in animals immunised with synthetic foot-and-mouth disease virus.

A good correlation exists between specific neutralising antibody titre and protection against challenge with foot-and-mouth disease virus (FMDV) in infected or virus-vaccinated cattle, but not in the case of animals immunised with synthetic FMDV peptides. Therefore, mechanisms other than simple neutralisation are likely to be important in vivo. Antibody affinity may influence the protective capacity of sera from immunised animals and experiments were carried out to measure the functional affinity for synthetic FMDV peptide of sera from guinea pigs and cattle given various synthetic vaccines. In guinea pigs given a single dose of synthetic vaccine, antibody affinity increased with time after immunisation. In cattle, however, administration of a second dose of peptide 21 days after the first markedly retarded the process of affinity maturation. For guinea pig sera of equivalent neutralising activity, those of higher functional affinity had higher protective indices than those of lower functional affinity. Knowledge of the importance of antibody affinity in protection against FMD is important for an improved understanding of the mechanisms of protection and for the design of novel vaccines.     

A CELL CULTURE VACCINE AGAINST BOVINE EPHEMERAL FEVER

SUMMARY A vaccine was prepared from cell culture fluids harvested from the twelfth passage of the 919 strain of bovine ephemeral fever (BEF) virus in Vero cell cultures. Cattle were vaccinated subcutaneously with various combinations of strain 919 virus and adjuvants. Neutralising antibodies were assayed at various times after vaccination and some cattle were challenged by intravenous inoculation with the virulent 417WBC strain of BEF virus. Strain 919 virus of the third and twelfth passage levels in Vero cells produced neither fever, clinical illness nor detectable viraemia in 5 calves inoculated intravenously. Nor could viraemia be detected in 5 heifers receiving vaccine subcutaneously. When the vaccine was administered mixed with aluminium hydroxide adjuvant, the production of neutralising antibodies increased with an increase in the volume of vaccine from 2.5 ml to 10 ml and the response to 2 injections was significantly better than the response to a single injection. The neutralising antibody response was decreased when vaccine was diluted in phosphate buffered saline. The neutralising antibody response following 2 subcutaneous vaccinations with strain 919 virus mixed with aluminium hydroxide adjuvant was higher than that following intravenous inoculation with virulent virus. The vaccine-induced antibodies persisted for at least 12 months, and revaccination at this time led to an increase in the titre of neutralising antibody. Antibodies induced by a single subcutaneous administration of strain 919 virus mixed with Freund's complete adjuvant persisted for at least 40 weeks; those induced by vaccine containing Freund's incomplete adjuvant had virtually disappeared within 16 weeks. All these calves responded to vaccination with aluminium hydroxide-containing vaccine with increases in levels of neutralising antibodies. Of 26 vaccinated calves challenged with virulent BEF virus, 24 remained clinically normal. Two developed brief periods of pyrexia on the seventh day after challenge, but no other clinical signs. One of these calves had a viraemia that was demonstrated only by intravenous inoculation of a susceptible calf. The remaining calf had no detectable viraemia. All of 7 unvaccinated calves developed severe clinical BEF within 5 days of challenge. No disease attributable to the 919 virus occurred in 24 vaccinated pregnant heifers or their newborn calves.     

Precipitating antibodies in experimental visna and natural progressive pneumonia of sheep

Serological responses of Icelandic sheep experimentally infected with visna virus (vv) were contrasted with responses in American Targhee sheep naturally infected with progressive pneumonia virus (PPV). Precipitating antibodies assayed by immunodiffusion were compared with the neutralising and complementing fixing antibody response. In experimental infections with vv, complement fixing and neutralising antibodies appeared early after infection and rose to high levels in all sheep, while precipitating antibodies were detected only at minimal titre. In natural infections with PPV, immune responses were less consistent and precipitating antibodies were detected more frequently than complement fixing or neutralising antibodies against PPV. These results may suggest important biological differences between the lytic fibroblast-tropic virus strains used for experimental infection of Icelandic sheep and the nonlytic macrophage-tropic strains of PPV circulating in nature. Lytic strains evoke a brisk response against the viral glycoprotein with high titre neutralising antibody while nonlytic strains induce a less consistent response to the glycoprotein.     

Intraocular vaccination with an inactivated highly pathogenic avian influenza virus induces protective antibody responses in chickens

Because it is expected to induce cross-reactive serum and mucosal antibody responses, mucosal vaccination against highly pathogenic avian influenza (HPAI) is potentially superior to conventional parenteral vaccination. Here, we tested whether intraocular vaccination with an inactivated AI virus induced protective antibody responses in chickens. Chickens were inoculated intraocularly twice with 10⁴ hemagglutination units of an inactivated H5N1 HPAI virus. Four weeks after the second vaccination, the chickens were challenged with a lethal dose of the homologous H5N1 HPAI virus. Results showed that most of the vaccinated chickens mounted positive antibody responses. The median serum hemagglutination inhibition titer was 1:80. Addition of CpG oligodeoxynucleotide 2006 or cholera toxin to the vaccine did not enhance serum antibody titers. Cross-reactive anti-hemagglutinin IgG, but not IgA, was detected in oropharyngeal secretions. In accordance with these antibody results, most vaccinated chickens survived a lethal challenge with the H5N1 HPAI virus and did not shed the challenge virus in respiratory or digestive tract secretions. Our results show that intraocular vaccination with an inactivated AI virus induces not only systemic but also mucosal antibody responses and confers protection against HPAI in chickens.     

Modulation of IgG subclass expression during antibody responses in sheep

Experiments were undertaken to investigate IgG subclass expression during epitope-specific antibody responses in sheep. Animals were immunised with conjugates of bovine serum albumin-DNP (BSA-DNP), or killed Staphylococcus aureus-DNP (Sa-DNP) alone or with muramyl dipeptide (MDP), dextran sulphate (DXS), or staphylococcal exotoxins (toxin). Sheep received two injections of the same preparation intracutaneously at six weeks interval. Total and IgG subclass-specific anti-DNP, and anti-carrier (BSA or S aureus) antibody levels were measured by ELISA in blood taken at weekly intervals before and after immunisation. Anti-DNP antibody levels in animals given Sa-DNP alone were considerably greater than in those immunised with BSA-DNP alone. Toxin suppressed antibody responses to DNP and both carriers; MDP suppressed anti-hapten antibody responses below the levels obtained with antigen alone. Neither toxin nor MDP significantly altered the IgG subclass profile of antibody to DNP bound to either carrier. DXS did not significantly change total levels of anti-DNP antibody measured in sheep given BSA-DNP or S aureus-DNP. However, DXS promoted IgG1 and suppressed IgG2 anti-DNP antibody responses during the secondary response to Sa-DNP but not to BSA-DNP.     

Humoral immunity in the ewe. 2. The effect of pregnancy on the primary and secondary antibody response to protein antigen

This study examines the effect of pregnancy on the quantity and isotype of antibody in ewes immunised with a novel primary antigen. The primary and secondary antibody levels to bovine serum albumin (BSA) in alum adjuvant, were compared between non-pregnant ewes and ewes immunised at different stages of pregnancy. Anti-BSA isotype specific responses were measured using an indirect ELISA. Results show the levels of immunoglobulin M (IgM) increased and persisted in response to BSA during pregnancy (P less than 0.05). Secondary immunoglobulin G1 (IgG1) titres were significantly impaired in late pregnancy and during lactation (P less than 0.05). The lower levels of immunoglobulin G2 (IgG2) were unaffected by pregnancy under these experimental conditions. Ewes were also immunised with BSA in alum adjuvant for their primary inoculum and BSA in different adjuvants for the secondary inoculation. Primary IgM persisted at higher levels during pregnancy compared with the response in non-pregnant ewes (P less than 0.05). Following the secondary injection, lower levels of anti-BSA specific IgM, IgG1 and IgG2 antibodies were produced in late pregnancy and during lactation compared with the levels in non-pregnant control ewes (P less than 0.05). Alterations in regulatory T-cell function or effector B-cell activity would most readily explain the qualitative changes in antibody titre observed following primary injection during pregnancy. The results also suggest an associated impairment of immunological memory following primary immunisation during pregnancy.     

Comparative immunogenicity of two bivalent botulinum vaccines

OBJECTIVE: To compare the ability of a new single-dose botulinum vaccine containing a non-mineral oil adjuvant with a single dose of a conventional botulinum vaccine product to produce antibody to Clostridium botulinum types C and D in cattle in Northern Australia. DESIGN AND PROCEDURE: One hundred and fifty Brahman steer weaners were randomly divided into two groups receiving either a single dose of CSL Bivalent Botulinum vaccine or Websters Singvac. Blood samples were collected at 0, 8 and 24 weeks and tested by antibody ELISA. The final samples were also tested by the toxin neutralisation test, to test titres of neutralising antibody. RESULTS: Six months after inoculation, cattle vaccinated with Websters Singvac had ELISA antibody response twice that of CSL conventional product. However, this difference was only evident for neutralising antibody to type C botulinum toxin. Both products produced similar titres of type D neutralising antibody after a single dose. CONCLUSION: Websters' Singvac produces a greater neutralising antibody response to type C botulism upon single inoculation than a conventional vaccine. The product produces an equivalent neutralising antibody response to type D.     

Antigen dose modulates the immunoglobulin isotype responses of pigs against intramuscularly administered F4-fimbriae

Parenteral immunisation normally induces a systemic antibody response characterised by high IgG and low IgA responses. In the present study, the effect of different doses of F4-fimbriae on the isotype-specific antibody response after intramuscular immunisation was studied in pigs. Pigs were injected twice with a 9 weeks interval with either 1, 0.1 or 0.01 mg of F4-ETEC fimbriae. The dose of 1mg F4 induced significantly lower primary F4-specific IgG and IgM responses than the doses of 0.1 and 0.01 mg F4, but primed for an enhanced F4-specific IgM serum antibody response after the booster immunisation. Furthermore, the dose of 0.1mg induced the highest F4-specific IgA serum response which was significantly higher than after injection with 0.01 and 1mg F4. Moreover, both lower doses (0.1 and 0.01 mg) showed a higher number of F4-specific IgA and IgG antibody secreting cells (ASC) in the local draining lymph nodes of the pigs. This study demonstrated that low doses of purified F4-ETEC fimbriae, especially the 0.1mg dose, are optimal for inducing F4-specific IgA responses after IM immunisation.     

Isotype-specific ELISAs for the detection of antibodies to bovine respiratory syncytial virus

Isotype-specific ELISAs for the detection of antibodies to bovine respiratory syncytial virus (BRSV) are described. BRSV-specific IgG1 and IgG2 were determined in indirect double antibody sandwich assays. For IgA and IgM antibody capture assays were used. The isotype specificity of the assays was confirmed by the observation that samples with a high titre of BRSV-specific antibodies of particular isotype were negative in the assays for the other isotypes and vice versa. Comparison of the results obtained in the ELISAs and in the virus neutralisation test showed that acute phase antibodies were more efficiently detected in the latter. It also showed that the presence of BRSV-specific IgA was not correlated with neutralising activity in vitro. The serum antibody response of BRSV-infected seronegative calves from the field consisted of a nearly simultaneous increase of IgM, IgA and IgG1-antibodies in the acute phase of the disease, while the IgG2-response followed at various intervals thereafter. In young animals with maternal antibodies a different pattern was found. There was no increase in IgG1 and IgG2, but six of eight animals showed a weak IgM response and two of these six calves also showed a weak and short lasting IgA response. Because maternal antibodies are insufficiently effective in protecting calves against BRSV, the presence of such antibodies at mucosal surfaces was investigated. Maternal immunity was found to be restricted to IgG1 antibodies in serum. This agrees with the failure of maternal antibodies to protect mucosal surfaces against BRSV infection.     

Priming for local and systemic antibody memory responses to bovine respiratory syncytial virus: effect of amount of virus, virus replication, route of administration and maternal antibodies

We studied the conditions under which calves can be primed for mucosal and serum antibody memory responses against bovine respiratory syncytial virus (BRSV), and the relationship between such responses and protection against the virus. Calves were primed via the respiratory tract with a low or high amount of live virus, with killed virus, or intramuscularly with live virus. Calves were challenged via the respiratory tract. Priming with live virus via the respiratory tract induced primary antibody responses in serum and on the mucosae, which were identical after the low and the high amount of virus. These responses were suppressed by maternal antibodies. Intramuscular priming of seronegative calves induced serum IgG1 and sometimes serum IgM and IgG2 responses, but no responses were detected on the mucosae. Sera of calves primed by the intramuscular or the respiratory route recognized the same viral proteins. No responses were observed after priming with killed virus, or after intramuscular priming of calves with maternal antibodies. After challenge, mucosal and serum antibody memory responses developed in calves that had been primed via the respiratory tract with live virus, whether they had maternal antibodies or not. One colostrum-fed calf showed a mucosal memory response, although serum responses were still suppressed by maternal antibodies. None of the calves thus primed shed virus after challenge. Intramuscular priming also primed for mucosal and serum memory responses after challenge, which however started perhaps slightly later and were not associated with protection against virus shedding. Priming with killed virus, or with live virus intramuscularly in the presence of maternal antibodies proved least effective in inducing memory and protection against virus shedding. Thus, protection against virus shedding was afforded by priming with live virus via the respiratory tract, both in calves with an without maternal antibodies. Protection was associated with a strong and rapid mucosal antibody memory response, but the reverse was not necessarily true. Protection against virus excretion had no relationship to titers of serum neutralizing or serum IgG1 or nasal IgA antibodies at the time of challenge.     

The effect of adjuvants on active and passive immunity in pregnant deer and their offspring.

Experiments were carried out to determine the effects of a range of adjuvants on immunoglobulin M (IgM) and immunoglobulin G (IgG) serum concentrations to a protein antigen administered subcutaneously to farmed female deer following mating. The antibody responses of animals immunised with keyhole limpet hemocyanin (KLH) in Freund's Incomplete Adjuvant (FIA), diethylaminoethyl dextran (DEAE-dextran) and aluminium hydroxide (alum) were compared with the response to antigen administered in the absence of adjuvants. Animals were subsequently challenged with a subcutaneous immunisation of the antigen in saline. Following parturition, the concentration of passively transferred antigen-specific antibody was measured in the serum of the offspring. The polyionic adjuvant, DEAE-dextran, produced the greatest enhancement of both primary and secondary IgG responses to KLH. Offspring suckling from mothers immunised with antigen in DEAE-dextran consequently had higher concentrations of specific antibodies in their serum than other fawns in the experiment. The adjuvants FIA and alum were approximately 20-fold less effective in enhancing antigen-specific IgG than DEAE-dextran but induced greater amounts of antigen-specific IgM. From the results presented in this paper, there is evidence that immunisation of deer during pregnancy may be an effective way of reducing morbidity in both mothers and offspring.     

Does multiple oral vaccination of wild boar against classical swine fever (CSF) have a positive influence on the immunity?

We studied the efficacy of multiple vaccinations of wild boar against classical swine fever (CSF) using a C-strain vaccine. The study consisted of two experiments. In the first experiment, 7 to 8 months old animals were vaccinated either three or four times at an interval of 7 days or twice at an interval of 14 or 28 days. In the second experiment, the efficacy of oral immunisation in young boars (3 months old) was examined after fivefold vaccination at intervals of 14 or 28 days. Independently of the immunisation scheme all wild boar developed neutralising antibodies. An evaluation of the antibody titres 28 days after the initial vaccine application showed that single vaccination and triple immunisation at an interval of 7 days induced the highest antibody titres (X > or = 1/80). In multiple vaccinated young boars (vaccinated at intervals of 14 or 28 days) the third vaccination led to a slight reduction or to an only moderate increase of the antibody titre. In a challenge study after the fifth vaccination all wild boar were protected (no viraemia, no virus excretion, no post-mortem virus detection in organs). This was confirmed by the fact that sentinel animals were not affected. Although other immunisation schemes also were effective, booster vaccination at an interval of 28 days is recommended as basic procedure for eradication of CSF in wild boar. Triple vaccination might also be used at the beginning of the control measures.