European Journal of Wildlife Research

, Volume 52, Issue 4, pp 271–276

Oral immunisation of wild boar against classical swine fever: uptake studies of new baits and investigations on the stability of lyophilised C-strain vaccine

Authors

  • A. Brauer
    • Institute of Infectology, Friedrich-Loeffler-InstitutFederal Research Institute for Animal Health
  • E. Lange
    • Institute of Infectology, Friedrich-Loeffler-InstitutFederal Research Institute for Animal Health
    • Institute of Infectology, Friedrich-Loeffler-InstitutFederal Research Institute for Animal Health
Original Paper

DOI: 10.1007/s10344-006-0048-0

Cite this article as:
Brauer, A., Lange, E. & Kaden, V. Eur J Wildl Res (2006) 52: 271. doi:10.1007/s10344-006-0048-0
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Abstract

The aim of this study was to investigate the uptake of new spherical and cuboid baits by wild boar and domestic pigs and to evaluate the stability of lyophilised C-strain vaccine stored at different environmental temperatures. New baits were designed to improve the consumption of the vaccine against classical swine fever by young wild boar. Our uptake studies showed that neither wild boar nor domestic pigs at the age of 2 months picked up the baits at all. Although the animals began to pick up the baits incompletely at the age of 3 months, a complete uptake of both the new baits and the older ones was only observed from the age of 4 (domestic pigs) and 4.5 (wild boar) months on. Nevertheless, the larger spherical baits with a diameter of 3 cm were taken up more effectively than the recent vaccine baits. As expected, lyophilised vaccine showed a higher stability than liquid vaccine, especially at temperatures ≥24°C. Independently of the stabiliser (GS4 or TSM) used, there are no differences between the virus titres detected within a storage period of 7 days. As the lyophilised C-strain vaccine was more stable at higher temperatures than the liquid vaccine formulation, we recommend to use lyophilised vaccine for oral immunisation of wild boar in the future, as oral vaccination is also carried out in the summertime.

Keywords

Classical swine feverWild boarOral immunisationNew baitsBait uptakeStability of vaccine

Introduction

Classical swine fever (CSF) is a highly contagious infectious disease of domestic pigs, European wild boar, and feral pigs caused by a pestivirus (Van Regenmortel et al. 2000). Outbreaks of CSF in domestic pigs result in economic damages not only in agriculture but also in other economic sectors and have negative consequences for animal welfare. If the disease is confirmed in wild boar, direct and indirect losses are also observed in forestry and agriculture (pig production), especially in the latter as a result of trade restrictions. As shown in Germany, oral immunisation of wild boar against CSF may be an effective measure within the control scenario (Kaden et al. 2000, 2002, 2003a; Kern and Lahrmann 2000). It was also carried out successfully in Luxembourg (Besch, personal communication), and oral vaccination of wild boar now is part of the control measures in France, in the Slovak Republic, and in Bulgaria. The goal of vaccination is to improve the herd immunity and to interrupt the chain of infection (Kaden et al. 2000, 2002, 2003a, 2005b). The recent oral vaccination procedure is based on a bait vaccine (Kaden and Lange 1998, 2001) and three double vaccinations per year (Kaden et al. 2003a, 2005b). In contrast, the vaccination procedure in the first field studies in the federal states Lower Saxony, Mecklenburg-Western Pomerania, and Brandenburg consisted of two double vaccination campaigns in spring and autumn (Kaden et al. 2000, 2002). Whereas the disease could be eradicated within a relatively short time in Lower Saxony (Kaden et al. 2000), vaccination in the other two countries had to be continued for a longer time period. Field studies revealed that the seroprevalence in young wild boar was significantly lower than in older animals. The low herd immunity in young animals may be one reason for the delayed CSF eradication in these countries. Based on the improved immunisation procedure consisting of three double vaccinations per year, higher seroprevalence rates were achieved in young animals as shown in Baden-Württenberg (Kaden et al. 2003a, 2005b) and in Rhineland-Palatinate (Kaden et al. 2003b). Despite this positive effect in young wild boar, the seroprevalence rates were still higher in older ones. Therefore, the development of smaller baits, which are better adapted to young animals, is the goal of the scientific work in a European project.

The first objective of this study was to analyse the uptake rate of newly created baits in domestic pigs and wild boar. The smaller baits should improve the consumption of the vaccine baits by young animals. The second aim was to investigate the stability of lyophilised C-strain vaccine.

Materials and methods

Vaccine

All experiments were based on the C-strain vaccine, which is used for oral vaccination of wild boar against CSF (Kaden and Lange 2001; Kaden et al. 2002). This vaccine, designated ‘RIEMSER Schweinepestoralvakzine’ (RIEMSER Arzneimittel AG, Greifswald-Insel Riems, Germany), is available in blisters in liquid form (about 1.6 ml vaccine per blister) and covered with a cereal-based bait matrix. The size of the baits is 4×4×1.5 cm (Fig. 1a). This liquid vaccine was used as control (reference vaccine) for all experiments carried out.
https://static-content.springer.com/image/art%3A10.1007%2Fs10344-006-0048-0/MediaObjects/10344_2006_48_Fig1_HTML.jpg
Fig. 1

Bait forms used for uptake studies

For the new baits, a different vaccine capsule and lyophilised vaccine form are necessary. Therefore, C-strain vaccine was mixed with stabilisers. The first vaccine formulation (vaccine A) contained the stabiliser ‘TSM,’ the second one the stabiliser ‘GS4’ (vaccine B). Both stabilisers were added to the liquid vaccine to a definite volume. For studies with the lyophilised C-strain vaccine, the stabilised vaccine was filled into sterilised glass vials (volume 1 ml), covered carefully with a rubber stopper, and then freeze-dried for 3 days. After lyophilisation the vials were closed with a plastic cap. Before and after the freeze-drying process samples were taken for sterility testing. This lyophilised vaccine was the basis for studies on the stability of the vaccine virus.

Creation of new baits

The recent vaccine bait (Fig. 1a) and a round one (size \( \emptyset \) 4×0.8 cm, Fig. 1b) previously investigated were included in the uptake studies. Different shapes and sizes of new baits were created for uptake studies based on hard fat maize matrix flavoured with almond aroma. Three bait forms were established, two spherical ones (diameters 1.8 and 3.0 cm, Fig. 1c) and one cuboid bait form (size 2.5×2.0×1.5 cm, Fig. 1d).

Uptake studies of new baits

Animals, housing, and feeding

Five wild boar at the age of 2 months derived from our own breeding unit and five weaner piglets obtained from a pig breeding farm in Mecklenburg-Western Pomerania were used for uptake studies. Domestic pigs were involved in this study because previous uptake experiments using the recent vaccine baits were mostly carried out with weaner piglets for want of young wild boar. The animals were kept conventionally on straw in a quarantine unit. They were fed with pellets twice a day, whereas water intake was possible ad libitum. The bait uptake was carried out in the stable.

Performance of the uptake study

The uptake study was started at the age of 2 months and continued at different intervals until the age of 6 months (for details, see Tables 1 and 2). Before laying out of the new bait forms maize was administered on the floor. Then several baits of all forms described above were laid out so that the animals had the opportunity to select between the different kinds of baits. For evaluation of the bait uptake, the behaviour of the animals during the uptake, the sequence of the bait uptake, and the uptake time were assessed or determined. The uptake was evaluated ranging from none (−) to very effective (+++):
  1. +

    Incomplete bait consumption: partial uptake of the bait matrix

     
  2. ++

    Complete bait consumption: most of the animals showed a complete but delayed bait uptake

     
  3. +++

    Complete bait consumption: all of the animals rapidly and completely picked up the baits

     
Table 1

Bait uptake by domestic pigs

Age (months)

Old baits

New baits

Spherical

Cuboid

Recent 4×4×1.5 cm

Round \( \emptyset \) 4×0.8 cm

\( \emptyset \) 1.8 cma

\( \emptyset \) 3.0 cm

2.5×2×1.5 cm

2

b

b

b

  

3

+

+

++

 

+

4

++

 

+++

++

++

5

++

 

+++c

+++

++

6

++

 

+++

+++

++

None, + incomplete bait consumption (partial uptake of the bait matrix), ++ complete bait consumption (most of the animals showed a complete but delayed bait uptake), and +++ complete bait consumption (all of the animals rapidly and completely picked up the baits)

aBaits consisted of hard fat/gelucire and maize as matrix

bNibbling, sniffling

cPicked up first

Table 2

Bait uptake by wild boar

Age (months)

Old baits

New baits

Spherical

Cuboid

Recent 4×4×1.5 cm

Round \( \emptyset \) 4×0.8 cm

\( \emptyset \) 1.8 cma

\( \emptyset \) 3.0 cm

2.5×2×1.5 cm

2

  

2.5

 

3.5

+

+

++

 

+

4.5

++

 

+++

++

++

5

++

 

+++

+++b

++

6

+++

 

+++

+++

+++

None, + incomplete bait consumption (partial uptake of the bait matrix), ++ complete bait consumption (most of the animals showed a complete but delayed bait uptake), and +++ complete bait consumption (all of the animals rapidly and completely picked up the baits)

aBaits consisted of hard fat/gelucire and maize as matrix

bPicked up first

Studies on the stability of lyophilised and liquid C-strain vaccine

Procedure

Until the beginning of the study the vials were stored in the refrigerator at 4°C. To evaluate the stability of the lyophilised vaccines (vaccines A and B) the vials were stored at different temperatures: 4°C (refrigerator), 13°C (soil), 24°C (room temperature), and 37°C (incubator). Two vials of each vaccine at different temperatures were collected daily for a period of 7 days. Liquid C-strain vaccine (control, reference vaccine) was subjected to the same procedure.

Virus titration

The re-suspended vaccine (1 ml sterile Aqua dest. per vial) was titrated on confluent EFN-R cells (CCLV, RIE 86) grown in 96-well cell culture plates. For this purpose, 100 μl of the log10 steps of the re-suspended vaccine dilutions was inoculated into four wells. After virus adsorption for 1 h at 37°C in a CO2-incubator 100 μl of cell culture medium was added per well. After an incubation period of 3 days at 37°C in a CO2-incubator (5% CO2) the medium was removed, the plates (wells) were washed with PBS (phosphate-buffered saline) and air-dried. After heat fixation at 80°C for 2 h the plates were stained for detection of CSF virus (CSFV). For this purpose, a mouse anti-CSFV-E2 mono-clonal antibody (provided by Bernd Köllner, Insel Riems) was used in combination with a horseradish peroxidase-conjugated anti-mouse antibody (Pierce, Rockford, USA).

Statistical analysis

The statistical evaluation of the stability of the C-strain vaccine was carried out with the statistic program SigmaStat 3.0 (SPSS Science Software Gmb, Erkrath, Germany) using the Mann–Whitney Rank Sum Test.

Results

Uptake studies of new bait forms

The studies on the bait uptake of wild boar and domestic pigs of different ages (Tables 1 and 2) demonstrate that neither wild boar piglets nor domestic pigs (weaner piglets) at the age of 2 months picked up the baits at all. The new spherical baits with a diameter of 1.8 cm and the new cuboid baits and the older ones were not picked up by 2.5-month-old wild boar. In contrast, domestic pigs began to take up baits at the age of 3 months. A moderate bait uptake was detected at this age for the small spherical baits, whereas the cuboid baits of the recent oral vaccine and the newly developed cuboid baits were only picked up incompletely. Moreover, 3.5-month-old wild boar piglets picked up all kinds of baits incompletely; the acceptance of the small spherical ones (diameter 1.8 cm) was higher. Most of these wild boar showed a complete bait consumption. From the age of 4 (domestic pigs) and 4.5 (wild boar) months the new baits and also the older ones were taken up well by both species. Assuming that the small spherical baits are the most suitable form for young gruntlings, the bait consumption was not studied completely for all bait forms.

Stability of the lyophilised vaccine formulations

The results of the studies on the stability of C-strain virus at different environmental conditions are summarised in Figs. 2, 3 and 4. As shown, the virus titre of the lyophilised vaccine was stable independent from the environmental temperatures and the kind of stabiliser used. Although the lowest titres of the freeze-dried vaccine were detected at 37°C in the incubator independent of the stabiliser added, there are no indications for a statistically significant difference between different days and temperatures. It seems that C-strain vaccine stabilised with TSM (Fig. 3) shows a continuous decrease of the virus titre at 37°C. However, statistical differences could not be found within the observation period. In contrast, liquid vaccine was not as stable as the lyophilised vaccine (Fig. 4). The virus titre already decreased at room temperature and at 37°C within three to 4 days.
https://static-content.springer.com/image/art%3A10.1007%2Fs10344-006-0048-0/MediaObjects/10344_2006_48_Fig2_HTML.gif
Fig. 2

Stability of C-strain vaccine freeze-dried with GS4 (mean virus titres and standard deviations)

https://static-content.springer.com/image/art%3A10.1007%2Fs10344-006-0048-0/MediaObjects/10344_2006_48_Fig3_HTML.gif
Fig. 3

Stability of C-strain vaccine freeze-dried with TSM (mean virus titres and standard deviations)

https://static-content.springer.com/image/art%3A10.1007%2Fs10344-006-0048-0/MediaObjects/10344_2006_48_Fig4_HTML.gif
Fig. 4

Stability of liquid C-strain vaccine (mean virus titres and standard deviations)

Discussion and conclusions

Although the immunity in wild boar could be improved after introduction of the triple or double vaccination against CSF (Kaden et al. 2002, 2003a, 2005a), the percentage of seropositive young wild boar is still relatively low, especially the proportion of seropositive gruntlings (animals ≤6 months of age). To improve the proportion of immunised gruntlings, there are theoretically two ways: (1) introduction of a prolonged maternal protection, and (2) bait uptake at an earlier age. As the first postulation cannot be fulfiled by vaccination (Kaden et al. 1999; Kaden and Lange 2004) the goal of our study was to create smaller baits to guarantee an earlier bait uptake of the young ones.

Three new bait forms were created based on the bait matrix used for the previous bait vaccine. Our uptake studies revealed that neither domestic pigs nor wild boar picked up the new baits effectively during the first 3 months of life independent of the size and the shape of the baits. These result indicate that for physiological reasons oral vaccination of young wild boar during the first 3 months of life is impossible. This confirms our previous experiences that gruntlings cannot be vaccinated actively during the first 3 months of life (Kaden 1999; Kaden et al. 1999, 2002, 2003a, 2005a). Whereas the wild boar piglets did not show any interest in the baits at the age of 2 months, the weaner piglets sniffed or nibbled at the baits. The slightly better uptake of the small spherical bait form by the 3-month-old domestic pigs and the 3.5-month-old wild boar demonstrates the beginning of an active bait uptake. The relatively late bait uptake of the wild boar piglets could be traced back to the fact that the gruntlings are still suckling during the first 3 months of life. Meynhardt (1984) reported that the young wild boar piglets stop suckling at the age of three to 4 months, i.e. at the time point when they come in contact with solid food. In contrast, domestic piglets receive solid food earlier. Although the domestic pigs picked up the baits slightly earlier and more effectively than wild boar, this does not explain the late bait uptake of these animals. The question whether it is possible to stimulate the uptake by feeding of placebo baits can only be answered speculatively. Nevertheless, an improved effectiveness of oral immunisation was reached by laying out placebo baits during the first field trial in Lower Saxony (Kaden et al. 2000). Hohne and Stone (1989), McIlroy et al. (1989), Saunders et al. (1990), Loepelmann and Dedek (1991), Loepelmann (1994), Schuster (1996) and others also reported higher uptake rates of baits by wildlife when placebos had been laid out before. An application of this procedure in field would be connected with a considerable additional workload, especially in the case of vaccination in enlarged areas. However, for physiological reasons it cannot be expected that the immunological gap in gruntlings up to the age of 3 to 4 months can be closed by such measures. Therefore, hunting of young wild boar (mainly gruntlings) and pregnant wild sows, which are not at the top of the sounder, is important for eradication of CSF.

The uptake studies in the stable showed that the spherical baits with a diameter of 3 cm were picked up more effectively than the ones recently used in field for vaccination of wild boar against CSF. Therefore, we recommend to use these baits in the future. However, this bait form requires a different vaccine capsule in connection with a lyophilised vaccine. Therefore, the stability of lyophilised C-strain vaccine was examined under different environmental conditions and temperatures. As expected, lyophilised C-strain vaccine proved to be more stable at higher temperatures (p<0.05 at 24 and 37°C) than liquid vaccine. Although the stability of the lyophilised C-strain vaccine with the anti-freeze GS4 seems to be slightly higher, this could not be statistically confirmed, except for storage at 24°C (p<0.05). Using both anti-freezes the virus titre was relatively constant within a temperature range from 4 to 24°C and for a storage period of 7 days. The virus titres were already slightly lower a few days after exposure to a temperature of 37°C in the incubator than at the other temperatures examined. It seems that the virus titre is more stable if the stabiliser GS4 is added. After storage for 7 days at 37°C the titre had decreased by 1 log10 from 105 to 104 tissue culture infective dose50 (TCID50)/ml. In the case of the TSM as stabiliser, the virus titre decreased from 105 to 103.5 TCID50/ml. These differences could not be statistically confirmed. However, the course of the titre of the C-strain virus stabilised with TSM shows a decreasing tendency when stored at 37°C for a longer time period. Therefore, GS4 should be favoured as stabiliser for lyophilisation. A rapid decrease of the virus titre was observed when liquid vaccine without a stabiliser was exposed to temperatures >20°C (Fig. 2). The virus titre already dropped after a short time of storage at 24°C from 105.5 TCID50/ml on day 0 to 103.5 TCID50/ml on day 4. A significant decrease (p<0.005) of the virus titre was also detected within a few days of storage in the incubator (37°C). No CSFV could be detected by virus isolation on day 6 of storage of the vaccine at 37°C. This result does not agree with previous investigations (Kaden, unpublished data). This study revealed that the liquid C-strain vaccine stabilised with TSM showed a relatively stable virus titre for 8 days when stored at temperatures between 0 and 20°C. Domestic pigs orally vaccinated with C-strain virus, which had been stored for 8 days at 20°C, were protected from an artificial infection with highly virulent CSFV. However, the virus titre decreased significantly within 4 days of storage at 37°C, and weaner pigs could not be immunised successfully when the vaccine was stored at this temperature for 8 days. The latter studies were carried out with the C-strain vaccine used in the first field trial in Lower Saxony (Kaden et al. 2000).

Our results indicate that lyophilised vaccine has a higher stability than liquid vaccine, especially under higher environmental temperatures. Therefore, it is recommended to use a new oral vaccine formulation for vaccination of wild boar against CSF based on new baits and a lyophilised vaccine. This vaccine formulation should be more effective for vaccination in summer, especially in countries with relatively high temperatures in the summertime.

Acknowledgements

We thank Malte Adelmann for his assistance in the preparation of the vaccine formulation; Dr. B. Lange, RIEMSER Arzneimittel AG, for kindly providing vaccine virus and vaccine stabilisers; and Jana Vesely for orienting us with the statistical analysis. We are also grateful to Anette Beidler for helpful discussion and critical reading of the manuscript. This study was supported by the EC project ‘CSF Vaccine & Wild Boar’ (contract number SSP 1-501599).

Copyright information

© Springer-Verlag 2006