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Oral vaccination of backyard pigs against classical swine fever
Veterinary Microbiology 163 (2013) 167–171
Contents lists available at SciVerse ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Short communication
Oral vaccination of backyard pigs against classical swine fever Vesna Milicevic a, Klaas Dietze b, Budimir Plavsic c, Mario Tikvicki d, Julio Pinto b, Klaus Depner e,* a
Institute of Veterinary Medicine of Serbia, Vojvode Totze 14, 1000 Belgrade, Serbia Food and Agriculture Organization of the UN (FAO), Viale delle Terme di Caracalla, 00153 Rome, Italy c Ministry of Agriculture, Forestry and Water Management, Veterinary Directorate, Omladinskih brigada 1, 11000 Belgrade, Serbia d Veterinary Clinic ‘‘Veterinar’’, Trg Paje Kujundzˇic´a 2/a, Subotica, Serbia e Friedrich-Loeffler-Institut, Su¨dufer 10, 17493 Greifswald-Insel Riems, Germany b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 13 September 2012 Received in revised form 27 November 2012 Accepted 6 December 2012
Vaccination represents a key pillar of most classical swine fever (CSF) control programmes. In backyard production systems the systematic implementation of parenteral vaccination proves to be difficult and alternative delivery mechanisms might be needed. In this study the use of oral vaccination against CSF in backyard pigs was tested under field conditions. The main objective was to assess uptake efficacy and immune response after oral vaccination of backyard pigs with bait vaccine currently used in the European Union for CSF control in wild boar (RIEMSER1 Schweinepestoralvakzine). In total 85 backyard pigs belonging to 16 holdings were vaccinated orally by distributing four baits per pig, given on two consecutive days. The baits were distributed on pen floors. 73% of the pigs older than 12 weeks (n = 41) had protective antibody titres 28 days after oral vaccination. The seroconversion rate in young piglets (n = 44, younger than 12 weeks) was 64%. Based on the results obtained we concluded that in an endemic situation where usual control measures (stamping out, zoning with restrictive measures, parenteral vaccination, etc.) may prove to be insufficient, a systematic vaccination based on the use of oral vaccine can be an appropriate tool for an improved CSF control in backyard production systems. ß 2012 Elsevier B.V. All rights reserved.
Keywords: Classical swine fever Oral vaccination Back yard pigs
1. Introduction On a global scale, classical swine fever (CSF) is considered one of the most important swine diseases with a significant effect on both, the profitability of commercial pig farming as well as on the livelihoods of farmers engaged in small-scale pig production leading to tremendous socio-economic consequences (Edwards et al., 2000). In several countries worldwide, CSF has been eradicated from the domestic pig population using mandatory vaccination in combination with sanitary measures (Van Oirschot, 2003). However, despite available tools to effectively control CSF, vaccination being the most
* Corresponding author. Tel.: +49 38351 71268; fax: +49 38351 71226. E-mail address: klaus.depner@fli.bund.de (K. Depner). 0378-1135/$ – see front matter ß 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetmic.2012.12.005
important one, many countries struggle in their efforts to reduce the CSF burden on the pig sector as large proportions of the global pig population are kept in diverse and rather unstructured small-scale production systems with low biosecurity and little interaction with veterinary services (FAO, 2011). In endemic CSF situations or in the crisis of a larger epidemic, where the usual control measures (stamping out, zoning, stand still, etc.) may prove to be insufficient, a systematic vaccination of the susceptible population is one of the most promising options for successful control and eradication, particularly in commercial and industrialized pig production systems. For pigs in less accessible production systems like backyard or free ranging systems, however, sustainable implementation of vaccination campaigns proved to be difficult and alternative approaches and delivery schemes might be needed.
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V. Milicevic et al. / Veterinary Microbiology 163 (2013) 167–171
The most widely used vaccines are based on the socalled ‘‘Chinese-strain’’ (C-strain) (Penrith et al., 2011). This live attenuated vaccine which is commonly regarded as the ‘‘gold standard’’ is highly efficacious after a single intramuscular application and induces long lasting immunity (Van Oirschot, 2003; Blome et al., 2006, 2012). The Cstrain vaccine has also been formulated as a bait vaccine for oral immunization of wild boar. In several experimental studies with domestic pigs as well as in field trials with wild boar it was shown that the oral C-strain vaccine is highly efficacious and safe (Kaden and Lange, 2001; Kaden et al., 2000, 2001, 2008, 2010). During the last decade oral vaccination of wild boar has been used successfully within the European Union (EU) representing an important pillar of the control strategy targeting a population where parenteral vaccine application is not practicable (Kaden et al., 2000, 2001, 2003, 2004; Kaden and Lange, 2001; von Ru¨den et al., 2008). In the presented study, the approach of using oral vaccination against CSF in backyard pigs was tested under field conditions. The main objective was to assess uptake efficacy and immune response after oral vaccination of backyard pigs with the commercially available C-strain bait vaccine currently used for CSF control in wild boar. 2. Materials and methods 2.1. Field trial design The study was conducted between April and May 2012 in two villages in the north of the Republic of Serbia and was integrated into the regular national CSF vaccination scheme. Farms that declared to have pigs to vaccinate at the time of the trial were incorporated randomly into the study. Data on sex, age, weight and the vaccination status of the sow was recorded. Only clinically healthy animals were included in the study. In total 16 holdings with a total of 85 pigs were selected according to the classification for ‘‘backyard holding’’ of the Serbian veterinary authorities (Anonymous, 2012). Backyard holdings are defined as holdings where pigs are kept mainly for own consumption and where biosecurity and hygiene measures are very poor and not efficient. One holding incorporated in the study had one pig, 11 holdings had between 2 and 5 pigs, 2 holdings had between 6 and 9 pigs and 3 holdings had between 10 and 15 pigs to vaccinate. In most of the cases, these pigs represented the total number of pigs on the farm. The youngest pigs were five weeks old while the oldest 21 weeks old. In total 44 pigs belonging to 8 farms were 12 weeks of age or younger (‘‘young piglets’’) while 41 pigs belonging to 11 farms were older than 12 weeks (‘‘old piglets’’), with 3 farms having animals in both age groups. The age classification was made on the assumption that piglets born by vaccinated sows might still have maternal antibodies if they are younger than 3 months while older piglets should be seronegative. According to the information given by the owners 52 of the 85 pigs were born by sows vaccinated against CSF in the previous years. In the group of young piglets, 24 piglets (55%) came from vaccinated sows and therefore the presence of maternal
antibodies was anticipated. For the remaining 20 piglets of that group the vaccination status of the mother sows could not be clarified. The pigs were kept the traditional way in small pig stables in close containment without contact to other pigs. The farmers were asked to group the animals that needed to be vaccinated in a separate pen and to not feed them in the morning before oral vaccination. For serological and virological examinations blood samples of all animals were taken on day 0 and on day 28 post-vaccination (dpv). 2.2. Vaccination scheme Commercially available and licensed RIEMSER1 Schweinepestoralvakzine was used according to the manufacturer’s recommendations (Riemser Arzneimittel AG, Greifswald-Insel Riems, Germany). The oral bait vaccine is made up by 1.6 ml C-strain ‘‘Riems’’ vaccine with a minimum titre of 104 TCID50 ml filled into a blister incorporated in a cereal-based bait matrix. It was calculated to vaccinate one pig with four baits: two baits administrated on day 0, the other two baits on day 1. The baits were distributed on the floor of the pens. Bait uptake was recorded on group basis since the uptake by individuals could not be monitored accurately when more than one pig was in the stable. For the period of 4 weeks post-vaccination owners were asked to report any abnormal behaviour or disease. 2.3. Laboratory investigations For antibody detection neutralization peroxidaselinked antibody assays (NPLA) were performed according to the diagnostic manual of the European Commission (Anonymous, 2002) using the following reference pestiviruses: CSF virus ‘‘Alfort-187’’/CSF0902/gt1.1, BVD virus ‘‘NADL’’ and BD virus ‘‘Moredun’’, kindly provided by the EU Reference Laboratory for CSF, Hannover, Germany. Antibody titres were calculated as neutralization doses 50 (ND50). Additionally to the NPLA a commercially available antibody enzyme-linked immunosorbent assay (ELISA) for the detection of CSF antibodies was used according to the protocol of the producer (PrioCHECK1 CSFV Ab 2.0, Prionics Lelystad B.V., The Netherlands). CSF viral RNA was detected by real time reverse transcription PCR (rt-RT-PCR) according to the protocol of Hoffmann et al. (2005) using primers derived from the 50 -nontranslated region. RNA extraction was carried out from whole blood by the QIAamp1 DNA Blood Mini Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions. 3. Results No adverse health events or side effects have been observed by the farmers and veterinarians throughout the observation period following the oral vaccine application. All pigs involved in the study showed interest in the baits when those were placed in the pen, yet the reaction and the overall uptake of baits differed between farms and
V. Milicevic et al. / Veterinary Microbiology 163 (2013) 167–171
169
Table 1 CSF vaccine bait uptake of back yard pigs of the two different age groups. Bait uptake
Group of young piglets (12 weeks)
Complete bait uptake of all baits on both days Complete bait uptake on one day, partial bait uptake on the other day Partial bait uptake on both days or partial uptake on one day and no uptake on other day
Group of older piglets (>12 weeks)
Pigs N = 44
Holdings
Pigs N = 41
Holdings
20 (45%)
3
31 (76%)
8
17 (39%)
3
5 (12%)
2
7 (16%)
2
5 (12%)
1
age groups. The bait uptake in the two different age groups is shown in Table 1. All pigs tested negative in the rt-RT-PCR on days 0 and 28 dpv. The detailed results of the NPLA for CSF are shown in Table 2. 73% of the older pigs (n = 41) had protective antibody titres 28 dpv. The seroconversion rate in young piglets (n = 44) was 64%. Neutralizing antibodies against ruminant pestiviruses were detected in 8 piglets 28 dpv. Five pigs had significant lower titres (3 times lower) against BVD virus than against CSF virus. Three pigs belonged to the category of older piglets and which tested seropositive for CSF at vaccination, had BVD titres which where higher or equal than the CSF titres. Antibodies against BD virus were not detected. All sera which were positive in the antibody ELISA 28 dpv were also positive in the neutralization test. However, 5 sera negative in the ELISA were found to be positive in the neutralization test with low titres (<40 ND50). 4. Discussion The main objective in the presented study was to assess under field conditions uptake efficacy and immune response of oral CSF bait vaccines in backyard pigs. So far, oral vaccination of domestic pigs against CSF has not been considered as a suitable disease control option (Suradhat et al., 2007). Oral vaccination against CSF has only been extensively used so far to control and eradicate
the disease from wild boar populations in Europe during the last decade (Kaden et al., 2002, 2003, 2004; von Ru¨den et al., 2008). The analysis of von Ru¨den et al. (2008) shows how a vaccination coverage of 69–76% in yearlings and adult wild boar was sufficient to eliminate CSF from the target population after 2 years of vaccination. Based on studies of Terpstra and Wensvoort (1988) we defined successful protection when the neutralizing antibody titres were above 40 ND50. In this field trial, 73% of the back yard pigs older than 12 weeks had protective antibody titres already after the first oral vaccination while the seroconversion rate in young piglets was lower (64%). The results clearly demonstrate that older pigs react much better immunologically than the younger ones and a vaccination coverage of above 70% can be achieved. The observed age dependence of the immune response to oral vaccine baits is in accordance with observations made with wild boar younger than 3 months (Brauer et al., 2006; Kaden and Lange, 2004; von Ru¨den et al., 2008). The lower rate of seroconversion in young piglets can be partially explained with the less complete bait uptake observed for this age group. The bait size represents a physical barrier and a complete uptake seems more ‘‘palatable’’ the older the pigs are. Additionally, the presence of maternal antibodies might have interfered with the vaccine virus as previously described by Suradhat et al. (2007). This is less likely to happen in pigs that passed the age of 12 weeks. Consequently, when aiming for high efficiency of oral vaccination schemes by using the commercial
Table 2 Neutralizing antibody titres before and after oral vaccination of piglets younger and older than 12 weeks. Pigs 12 weeks and younger (N = 44)
Virus neutralization titre development after vaccination
Titre at vaccination (ND50) 7 3
5 5
6
10–30
2 15 3 6
5 5 5 10–160
2
40–80
a
Pigs older than 12 weeks (N = 41)
Titre 28 days post vac. (ND50)
Interpretation
5 10–30
No immune reaction Slight seroconversion, inadequate protectiona No titre increase, inadequate protection Seroconversion, protection Seroconversion, protection Seroconversion, protection Significant titre increase, protection No titre increase, protection
5–30 40–80 120–640 >640 More than 3 increase 80
Inadequate protection was considered to be a titre below 40 ND50.
7 3 1 5 18 4 3 0
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V. Milicevic et al. / Veterinary Microbiology 163 (2013) 167–171
‘‘Riemser’’-vaccine the target population should comprise animals older than 12 weeks of age only. Although it was not evident if the reason for the lower seroconversion rate in the group of younger piglets was due to the presence of maternal antibodies, or the consequence of inappropriate baits, a formulation of oral bait vaccines of a smaller size, which might be better accepted by younger piglets, should be attempted. Four animals in the group of older piglets had CSF antibodies at the time of vaccination. It is unlikely that these antibodies were of maternal origin. Serological crossreactivity with ruminant petiviruses could not be excluded in 3 cases as these animals had positive BVD titres. The 4th animals status remains inconclusive, but since it belonged to the oldest animals in the trial (age: 21 weeks) a prior, not properly recorded vaccination against CSF could have taken place. Five sera taken 28 dpv tested negative in the ELISA and positive with low titres in the neutralization test. This discrepancy between the different serological tests can be attributed to the higher sensitivity of the neutralization test (Anonymous, 2002). Comparing oral vaccination of back yard pigs with that of wild boar, vaccine application is by far easier and more reliable. Since domestic pigs are usually confined in stables and their number is known, bait distribution according to the number of pigs can be performed easily by placing the baits on the floor, best before the pigs are fed. Theoretically one vaccine bait is sufficient to induce full protective immunity. If several pigs are in one stable it might happen that some pigs are eating more than one bait while others none. To minimize this risk pigs were vaccinated on two consecutive days calculating 4 baits per pig in total, increasing the bait uptake probability for each pig. This approach can be modified by minimizing the number of baits per pig if a controlled bait uptake can be assured (e.g. in stables with only one pig or individual feeding devices). As a non-invasive vaccine application the described method brings additional advantages in the field of biosecurity. Vaccination campaigns for small-scale pig producers often come along with an increased risk of disease transmission between farms. Due to the low number of animals per farm, vaccination teams normally have to visit plenty of farms in one day and would need to follow strict biosecurity protocols to avoid the spread of pathogens, sometimes simply not feasible due to resource constraints. Oral CSF vaccination does not require direct contact with the animals and depending on the local circumstances even the entry onto the farm premises by any visitor can be avoided as the vaccination can be performed by the farmer. From a logistical point of view, oral vaccination of domestic pigs in remote areas has the advantage of facilitated on-farm delivery. In experimental studies it could be shown that the vaccine keeps is efficiency for 3–4 days at room temperature (Brauer et al., 2006). Therefore the bait vaccine gives enough flexibility in the final delivery step from the local provider to the farm/ animal. Based on the obtained results we can conclude, that in an endemic situation for CSF where the usual control measures (stamping out, zoning with restrictive measures,
parenteral vaccination, etc.) may prove to be insufficient, a systematic vaccination of back yard pigs based on the use of oral vaccines can be an complementary tool in the efforts to control and eradicate CSF. Once potent live attenuated CSF marker vaccines will be available, the oral vaccine approach could be used also in a DIVA concept which would give further advantages in the fight against CSF. Conflict of interest statement The authors affirm that no financial or personal relationship existed that could have inappropriately influenced the content of this manuscript or the opinions expressed. Acknowledgements The authors wish to thank all persons in the field in Serbia involved in the oral vaccination trial as well as Wolfgang Bo¨hle and Anja Globig from the International Animal Health Team at the FLI for all the support given throughout the project. This study was supported through a Letter of Agreement signed between FAO and FLI. References Anonymous, 2002. Commission Decision of February 2002 approving a diagnostic manual establishing diagnostic procedures, sampling methods and criteria for evaluation of the laboratory tests for the confirmation of classical swine fever (2002/106/EC), Chapter VII. Off. J. Eur. Union L039, 71–88 (09.02.02). Anonymous, 2012. Annual program of animal health care measures for 2012. Republic of Serbia, Official Gazette No. 21/12. Blome, S., Meindl-Bo¨hmer, A., Loeffen, W., Thuer, B., Moennig, V., 2006. Assessment of classical swine fever diagnostics and vaccine performance. Rev. Sci. Tech. Off. Int. Epiz. 25 (3), 1025–1038. Blome, S., Aebischer, A., Lange, E., Hofmann, M., Leifer, I., Loeffen, W., Koenen, F., Beer, M., 2012. Comparative evaluation of live marker veccine candidates ‘‘CP7_E2als’’ and ‘‘flc11’’ along with C-strain ‘‘Riems’’ after oral vaccination. Vet. Microbiol. 158, 42–59. Brauer, A., Lange, E., Kaden, V., 2006. Oral immunization of wild boar against classical swine fever: uptake studies of new baits and investigations on the stability of lyophilized C-strain vaccine. Eur. J. Wildl. Res. 52, 271–276. Edwards, S., Fukusho, A., Lefevre, P., Lipowski, A., Pejsak, Z., Roehe, P., Westergaard, J., 2000. Classical swine fever: the global situation. Vet. Microbiol. 73 (2–3), 103–119. FAO, 2011. Overview of classical swine fever: learning from regional disease control strategies. By Pinto, J., Depner, K., Vargas-Tera´n, M. EMPRES Transboundary Animal Disease Bulletin No. 39, pp. 46–51. Hoffmann, B., Beer, M., Schelp, C., Schirrmeier, H., Depner, K., 2005. Validation of a real-time RT-PCR assay for sensitive and specific detection of classical swine fever. J. Vir. Meth. 130, 36–44. Kaden, V., Lange, E., Fischer, U., Strebelow, G., 2000. Oral immunisation of wild boar against classical swine fever: evaluation of the first field study in Germany. Vet. Microbiol. 73, 239–252. Kaden, V., Lange, B., 2001. Oral immunisation against classical swine fever (CSF): onset and duration of immunity. Vet. Microbiol. 82, 301–310. Kaden, V., Schurig, U., Steyer, H., 2001. Oral immunization of pigs against classical swine fever. Course of the disease and virus transmission after simultaneous vaccination and infection. Acta Virol. 45, 23–29. Kaden, V., Heyne, H., Kiupel, H., Letz, W., Kern, B., Lemmer, U., Gossger, K., Rothe, A., Bo¨hme, H., Tyrpe, P., 2002. Oral immunisation of wild boar against classical swine fever: concluding analysis of the recent field trials in Germany. Berl. Munch. Tierarztl. Wochenschr. 115, 179–185. Kaden, V., Renner, C., Rothe, A., Lange, E., Ha¨nel, A., Gossger, K., 2003. Evaluation of the oral immunisation of wild boar against classical swine fever in Baden-Wu¨rttemberg. Berl. Munch. Tierarztl. Wochenschr. 116, 362–367.
V. Milicevic et al. / Veterinary Microbiology 163 (2013) 167–171 Kaden, V., Lange, E., Steyer, H., 2004. Does multiple oral vaccination of wild boar against classical swine fever (CSF) have a positive influence on the immunity? Dtsch. Tierarztl. Wochenschr. 111, 63–67. Kaden, V., Lange, E., Steyer, H., Lange, B., Klopfleisch, R., Teifke, J.P., Bruer, W., 2008. Classical swine fever virus strain ‘‘C’’ protects the offspring by oral immunization of pregnant sows. Vet. Microbiol. 130, 20–27. Kaden, V., Lange, E., Ku¨ster, H., Mu¨ller, T., Lange, B., 2010. An update on safety studies on the attenuated ‘‘RIEMSER1 Schweinepestoralvakzine’’ for vaccination of wild boar against classical swine fever. Vet. Microbiol. 143, 133–138. Kaden, V., Lange, E., 2004. Development of maternal antibodies after oral vaccination of young female wild boar against classical swine fever. Vet. Microbiol. 103, 115–119.
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Penrith, M.L., Vosloo, W., et al., 2011. Classical swine fever (hog cholera): review of aspects relevant to control. Transbound. Emerg. Dis. 58 (3), 187–196. Suradhat, S., Damrongwatanapokin, S., Thanawongnuwech, R., 2007. Factor critical for successful vaccination against classical swine fever in endemic areas. Vet. Microbiol. 119, 1–9. Terpstra, C., Wensvoort, G., 1988. The protective value of vaccine-induced neutralising antibody titres in swine fever. Vet. Microbiol. 16, 123–128. Van Oirschot, J.T., 2003. Vaccinology of classical swine fever: from lab to field. Vet. Microbiol. 96 (4), 367–384. von Ru¨den, S., Staubach, C., Kaden, V., Hess, R.G., Blicke, J., Ku¨hne, S., Sonnenburg, J., Fro¨hlich, A., Teuffert, J., Moennig, V., 2008. Retrospective analysis of the oral immunisation of wild boar populations against classical swine fever virus (CSFV) in region Eifel of Rhineland-Palatinate. Vet. Microbiol. 132, 29–38.
Contents lists available at SciVerse ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Short communication
Oral vaccination of backyard pigs against classical swine fever Vesna Milicevic a, Klaas Dietze b, Budimir Plavsic c, Mario Tikvicki d, Julio Pinto b, Klaus Depner e,* a
Institute of Veterinary Medicine of Serbia, Vojvode Totze 14, 1000 Belgrade, Serbia Food and Agriculture Organization of the UN (FAO), Viale delle Terme di Caracalla, 00153 Rome, Italy c Ministry of Agriculture, Forestry and Water Management, Veterinary Directorate, Omladinskih brigada 1, 11000 Belgrade, Serbia d Veterinary Clinic ‘‘Veterinar’’, Trg Paje Kujundzˇic´a 2/a, Subotica, Serbia e Friedrich-Loeffler-Institut, Su¨dufer 10, 17493 Greifswald-Insel Riems, Germany b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 13 September 2012 Received in revised form 27 November 2012 Accepted 6 December 2012
Vaccination represents a key pillar of most classical swine fever (CSF) control programmes. In backyard production systems the systematic implementation of parenteral vaccination proves to be difficult and alternative delivery mechanisms might be needed. In this study the use of oral vaccination against CSF in backyard pigs was tested under field conditions. The main objective was to assess uptake efficacy and immune response after oral vaccination of backyard pigs with bait vaccine currently used in the European Union for CSF control in wild boar (RIEMSER1 Schweinepestoralvakzine). In total 85 backyard pigs belonging to 16 holdings were vaccinated orally by distributing four baits per pig, given on two consecutive days. The baits were distributed on pen floors. 73% of the pigs older than 12 weeks (n = 41) had protective antibody titres 28 days after oral vaccination. The seroconversion rate in young piglets (n = 44, younger than 12 weeks) was 64%. Based on the results obtained we concluded that in an endemic situation where usual control measures (stamping out, zoning with restrictive measures, parenteral vaccination, etc.) may prove to be insufficient, a systematic vaccination based on the use of oral vaccine can be an appropriate tool for an improved CSF control in backyard production systems. ß 2012 Elsevier B.V. All rights reserved.
Keywords: Classical swine fever Oral vaccination Back yard pigs
1. Introduction On a global scale, classical swine fever (CSF) is considered one of the most important swine diseases with a significant effect on both, the profitability of commercial pig farming as well as on the livelihoods of farmers engaged in small-scale pig production leading to tremendous socio-economic consequences (Edwards et al., 2000). In several countries worldwide, CSF has been eradicated from the domestic pig population using mandatory vaccination in combination with sanitary measures (Van Oirschot, 2003). However, despite available tools to effectively control CSF, vaccination being the most
* Corresponding author. Tel.: +49 38351 71268; fax: +49 38351 71226. E-mail address: klaus.depner@fli.bund.de (K. Depner). 0378-1135/$ – see front matter ß 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetmic.2012.12.005
important one, many countries struggle in their efforts to reduce the CSF burden on the pig sector as large proportions of the global pig population are kept in diverse and rather unstructured small-scale production systems with low biosecurity and little interaction with veterinary services (FAO, 2011). In endemic CSF situations or in the crisis of a larger epidemic, where the usual control measures (stamping out, zoning, stand still, etc.) may prove to be insufficient, a systematic vaccination of the susceptible population is one of the most promising options for successful control and eradication, particularly in commercial and industrialized pig production systems. For pigs in less accessible production systems like backyard or free ranging systems, however, sustainable implementation of vaccination campaigns proved to be difficult and alternative approaches and delivery schemes might be needed.
168
V. Milicevic et al. / Veterinary Microbiology 163 (2013) 167–171
The most widely used vaccines are based on the socalled ‘‘Chinese-strain’’ (C-strain) (Penrith et al., 2011). This live attenuated vaccine which is commonly regarded as the ‘‘gold standard’’ is highly efficacious after a single intramuscular application and induces long lasting immunity (Van Oirschot, 2003; Blome et al., 2006, 2012). The Cstrain vaccine has also been formulated as a bait vaccine for oral immunization of wild boar. In several experimental studies with domestic pigs as well as in field trials with wild boar it was shown that the oral C-strain vaccine is highly efficacious and safe (Kaden and Lange, 2001; Kaden et al., 2000, 2001, 2008, 2010). During the last decade oral vaccination of wild boar has been used successfully within the European Union (EU) representing an important pillar of the control strategy targeting a population where parenteral vaccine application is not practicable (Kaden et al., 2000, 2001, 2003, 2004; Kaden and Lange, 2001; von Ru¨den et al., 2008). In the presented study, the approach of using oral vaccination against CSF in backyard pigs was tested under field conditions. The main objective was to assess uptake efficacy and immune response after oral vaccination of backyard pigs with the commercially available C-strain bait vaccine currently used for CSF control in wild boar. 2. Materials and methods 2.1. Field trial design The study was conducted between April and May 2012 in two villages in the north of the Republic of Serbia and was integrated into the regular national CSF vaccination scheme. Farms that declared to have pigs to vaccinate at the time of the trial were incorporated randomly into the study. Data on sex, age, weight and the vaccination status of the sow was recorded. Only clinically healthy animals were included in the study. In total 16 holdings with a total of 85 pigs were selected according to the classification for ‘‘backyard holding’’ of the Serbian veterinary authorities (Anonymous, 2012). Backyard holdings are defined as holdings where pigs are kept mainly for own consumption and where biosecurity and hygiene measures are very poor and not efficient. One holding incorporated in the study had one pig, 11 holdings had between 2 and 5 pigs, 2 holdings had between 6 and 9 pigs and 3 holdings had between 10 and 15 pigs to vaccinate. In most of the cases, these pigs represented the total number of pigs on the farm. The youngest pigs were five weeks old while the oldest 21 weeks old. In total 44 pigs belonging to 8 farms were 12 weeks of age or younger (‘‘young piglets’’) while 41 pigs belonging to 11 farms were older than 12 weeks (‘‘old piglets’’), with 3 farms having animals in both age groups. The age classification was made on the assumption that piglets born by vaccinated sows might still have maternal antibodies if they are younger than 3 months while older piglets should be seronegative. According to the information given by the owners 52 of the 85 pigs were born by sows vaccinated against CSF in the previous years. In the group of young piglets, 24 piglets (55%) came from vaccinated sows and therefore the presence of maternal
antibodies was anticipated. For the remaining 20 piglets of that group the vaccination status of the mother sows could not be clarified. The pigs were kept the traditional way in small pig stables in close containment without contact to other pigs. The farmers were asked to group the animals that needed to be vaccinated in a separate pen and to not feed them in the morning before oral vaccination. For serological and virological examinations blood samples of all animals were taken on day 0 and on day 28 post-vaccination (dpv). 2.2. Vaccination scheme Commercially available and licensed RIEMSER1 Schweinepestoralvakzine was used according to the manufacturer’s recommendations (Riemser Arzneimittel AG, Greifswald-Insel Riems, Germany). The oral bait vaccine is made up by 1.6 ml C-strain ‘‘Riems’’ vaccine with a minimum titre of 104 TCID50 ml filled into a blister incorporated in a cereal-based bait matrix. It was calculated to vaccinate one pig with four baits: two baits administrated on day 0, the other two baits on day 1. The baits were distributed on the floor of the pens. Bait uptake was recorded on group basis since the uptake by individuals could not be monitored accurately when more than one pig was in the stable. For the period of 4 weeks post-vaccination owners were asked to report any abnormal behaviour or disease. 2.3. Laboratory investigations For antibody detection neutralization peroxidaselinked antibody assays (NPLA) were performed according to the diagnostic manual of the European Commission (Anonymous, 2002) using the following reference pestiviruses: CSF virus ‘‘Alfort-187’’/CSF0902/gt1.1, BVD virus ‘‘NADL’’ and BD virus ‘‘Moredun’’, kindly provided by the EU Reference Laboratory for CSF, Hannover, Germany. Antibody titres were calculated as neutralization doses 50 (ND50). Additionally to the NPLA a commercially available antibody enzyme-linked immunosorbent assay (ELISA) for the detection of CSF antibodies was used according to the protocol of the producer (PrioCHECK1 CSFV Ab 2.0, Prionics Lelystad B.V., The Netherlands). CSF viral RNA was detected by real time reverse transcription PCR (rt-RT-PCR) according to the protocol of Hoffmann et al. (2005) using primers derived from the 50 -nontranslated region. RNA extraction was carried out from whole blood by the QIAamp1 DNA Blood Mini Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions. 3. Results No adverse health events or side effects have been observed by the farmers and veterinarians throughout the observation period following the oral vaccine application. All pigs involved in the study showed interest in the baits when those were placed in the pen, yet the reaction and the overall uptake of baits differed between farms and
V. Milicevic et al. / Veterinary Microbiology 163 (2013) 167–171
169
Table 1 CSF vaccine bait uptake of back yard pigs of the two different age groups. Bait uptake
Group of young piglets (12 weeks)
Complete bait uptake of all baits on both days Complete bait uptake on one day, partial bait uptake on the other day Partial bait uptake on both days or partial uptake on one day and no uptake on other day
Group of older piglets (>12 weeks)
Pigs N = 44
Holdings
Pigs N = 41
Holdings
20 (45%)
3
31 (76%)
8
17 (39%)
3
5 (12%)
2
7 (16%)
2
5 (12%)
1
age groups. The bait uptake in the two different age groups is shown in Table 1. All pigs tested negative in the rt-RT-PCR on days 0 and 28 dpv. The detailed results of the NPLA for CSF are shown in Table 2. 73% of the older pigs (n = 41) had protective antibody titres 28 dpv. The seroconversion rate in young piglets (n = 44) was 64%. Neutralizing antibodies against ruminant pestiviruses were detected in 8 piglets 28 dpv. Five pigs had significant lower titres (3 times lower) against BVD virus than against CSF virus. Three pigs belonged to the category of older piglets and which tested seropositive for CSF at vaccination, had BVD titres which where higher or equal than the CSF titres. Antibodies against BD virus were not detected. All sera which were positive in the antibody ELISA 28 dpv were also positive in the neutralization test. However, 5 sera negative in the ELISA were found to be positive in the neutralization test with low titres (<40 ND50). 4. Discussion The main objective in the presented study was to assess under field conditions uptake efficacy and immune response of oral CSF bait vaccines in backyard pigs. So far, oral vaccination of domestic pigs against CSF has not been considered as a suitable disease control option (Suradhat et al., 2007). Oral vaccination against CSF has only been extensively used so far to control and eradicate
the disease from wild boar populations in Europe during the last decade (Kaden et al., 2002, 2003, 2004; von Ru¨den et al., 2008). The analysis of von Ru¨den et al. (2008) shows how a vaccination coverage of 69–76% in yearlings and adult wild boar was sufficient to eliminate CSF from the target population after 2 years of vaccination. Based on studies of Terpstra and Wensvoort (1988) we defined successful protection when the neutralizing antibody titres were above 40 ND50. In this field trial, 73% of the back yard pigs older than 12 weeks had protective antibody titres already after the first oral vaccination while the seroconversion rate in young piglets was lower (64%). The results clearly demonstrate that older pigs react much better immunologically than the younger ones and a vaccination coverage of above 70% can be achieved. The observed age dependence of the immune response to oral vaccine baits is in accordance with observations made with wild boar younger than 3 months (Brauer et al., 2006; Kaden and Lange, 2004; von Ru¨den et al., 2008). The lower rate of seroconversion in young piglets can be partially explained with the less complete bait uptake observed for this age group. The bait size represents a physical barrier and a complete uptake seems more ‘‘palatable’’ the older the pigs are. Additionally, the presence of maternal antibodies might have interfered with the vaccine virus as previously described by Suradhat et al. (2007). This is less likely to happen in pigs that passed the age of 12 weeks. Consequently, when aiming for high efficiency of oral vaccination schemes by using the commercial
Table 2 Neutralizing antibody titres before and after oral vaccination of piglets younger and older than 12 weeks. Pigs 12 weeks and younger (N = 44)
Virus neutralization titre development after vaccination
Titre at vaccination (ND50) 7 3
5 5
6
10–30
2 15 3 6
5 5 5 10–160
2
40–80
a
Pigs older than 12 weeks (N = 41)
Titre 28 days post vac. (ND50)
Interpretation
5 10–30
No immune reaction Slight seroconversion, inadequate protectiona No titre increase, inadequate protection Seroconversion, protection Seroconversion, protection Seroconversion, protection Significant titre increase, protection No titre increase, protection
5–30 40–80 120–640 >640 More than 3 increase 80
Inadequate protection was considered to be a titre below 40 ND50.
7 3 1 5 18 4 3 0
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‘‘Riemser’’-vaccine the target population should comprise animals older than 12 weeks of age only. Although it was not evident if the reason for the lower seroconversion rate in the group of younger piglets was due to the presence of maternal antibodies, or the consequence of inappropriate baits, a formulation of oral bait vaccines of a smaller size, which might be better accepted by younger piglets, should be attempted. Four animals in the group of older piglets had CSF antibodies at the time of vaccination. It is unlikely that these antibodies were of maternal origin. Serological crossreactivity with ruminant petiviruses could not be excluded in 3 cases as these animals had positive BVD titres. The 4th animals status remains inconclusive, but since it belonged to the oldest animals in the trial (age: 21 weeks) a prior, not properly recorded vaccination against CSF could have taken place. Five sera taken 28 dpv tested negative in the ELISA and positive with low titres in the neutralization test. This discrepancy between the different serological tests can be attributed to the higher sensitivity of the neutralization test (Anonymous, 2002). Comparing oral vaccination of back yard pigs with that of wild boar, vaccine application is by far easier and more reliable. Since domestic pigs are usually confined in stables and their number is known, bait distribution according to the number of pigs can be performed easily by placing the baits on the floor, best before the pigs are fed. Theoretically one vaccine bait is sufficient to induce full protective immunity. If several pigs are in one stable it might happen that some pigs are eating more than one bait while others none. To minimize this risk pigs were vaccinated on two consecutive days calculating 4 baits per pig in total, increasing the bait uptake probability for each pig. This approach can be modified by minimizing the number of baits per pig if a controlled bait uptake can be assured (e.g. in stables with only one pig or individual feeding devices). As a non-invasive vaccine application the described method brings additional advantages in the field of biosecurity. Vaccination campaigns for small-scale pig producers often come along with an increased risk of disease transmission between farms. Due to the low number of animals per farm, vaccination teams normally have to visit plenty of farms in one day and would need to follow strict biosecurity protocols to avoid the spread of pathogens, sometimes simply not feasible due to resource constraints. Oral CSF vaccination does not require direct contact with the animals and depending on the local circumstances even the entry onto the farm premises by any visitor can be avoided as the vaccination can be performed by the farmer. From a logistical point of view, oral vaccination of domestic pigs in remote areas has the advantage of facilitated on-farm delivery. In experimental studies it could be shown that the vaccine keeps is efficiency for 3–4 days at room temperature (Brauer et al., 2006). Therefore the bait vaccine gives enough flexibility in the final delivery step from the local provider to the farm/ animal. Based on the obtained results we can conclude, that in an endemic situation for CSF where the usual control measures (stamping out, zoning with restrictive measures,
parenteral vaccination, etc.) may prove to be insufficient, a systematic vaccination of back yard pigs based on the use of oral vaccines can be an complementary tool in the efforts to control and eradicate CSF. Once potent live attenuated CSF marker vaccines will be available, the oral vaccine approach could be used also in a DIVA concept which would give further advantages in the fight against CSF. Conflict of interest statement The authors affirm that no financial or personal relationship existed that could have inappropriately influenced the content of this manuscript or the opinions expressed. Acknowledgements The authors wish to thank all persons in the field in Serbia involved in the oral vaccination trial as well as Wolfgang Bo¨hle and Anja Globig from the International Animal Health Team at the FLI for all the support given throughout the project. This study was supported through a Letter of Agreement signed between FAO and FLI. References Anonymous, 2002. Commission Decision of February 2002 approving a diagnostic manual establishing diagnostic procedures, sampling methods and criteria for evaluation of the laboratory tests for the confirmation of classical swine fever (2002/106/EC), Chapter VII. Off. J. Eur. Union L039, 71–88 (09.02.02). Anonymous, 2012. Annual program of animal health care measures for 2012. Republic of Serbia, Official Gazette No. 21/12. Blome, S., Meindl-Bo¨hmer, A., Loeffen, W., Thuer, B., Moennig, V., 2006. Assessment of classical swine fever diagnostics and vaccine performance. Rev. Sci. Tech. Off. Int. Epiz. 25 (3), 1025–1038. Blome, S., Aebischer, A., Lange, E., Hofmann, M., Leifer, I., Loeffen, W., Koenen, F., Beer, M., 2012. Comparative evaluation of live marker veccine candidates ‘‘CP7_E2als’’ and ‘‘flc11’’ along with C-strain ‘‘Riems’’ after oral vaccination. Vet. Microbiol. 158, 42–59. Brauer, A., Lange, E., Kaden, V., 2006. Oral immunization of wild boar against classical swine fever: uptake studies of new baits and investigations on the stability of lyophilized C-strain vaccine. Eur. J. Wildl. Res. 52, 271–276. Edwards, S., Fukusho, A., Lefevre, P., Lipowski, A., Pejsak, Z., Roehe, P., Westergaard, J., 2000. Classical swine fever: the global situation. Vet. Microbiol. 73 (2–3), 103–119. FAO, 2011. Overview of classical swine fever: learning from regional disease control strategies. By Pinto, J., Depner, K., Vargas-Tera´n, M. EMPRES Transboundary Animal Disease Bulletin No. 39, pp. 46–51. Hoffmann, B., Beer, M., Schelp, C., Schirrmeier, H., Depner, K., 2005. Validation of a real-time RT-PCR assay for sensitive and specific detection of classical swine fever. J. Vir. Meth. 130, 36–44. Kaden, V., Lange, E., Fischer, U., Strebelow, G., 2000. Oral immunisation of wild boar against classical swine fever: evaluation of the first field study in Germany. Vet. Microbiol. 73, 239–252. Kaden, V., Lange, B., 2001. Oral immunisation against classical swine fever (CSF): onset and duration of immunity. Vet. Microbiol. 82, 301–310. Kaden, V., Schurig, U., Steyer, H., 2001. Oral immunization of pigs against classical swine fever. Course of the disease and virus transmission after simultaneous vaccination and infection. Acta Virol. 45, 23–29. Kaden, V., Heyne, H., Kiupel, H., Letz, W., Kern, B., Lemmer, U., Gossger, K., Rothe, A., Bo¨hme, H., Tyrpe, P., 2002. Oral immunisation of wild boar against classical swine fever: concluding analysis of the recent field trials in Germany. Berl. Munch. Tierarztl. Wochenschr. 115, 179–185. Kaden, V., Renner, C., Rothe, A., Lange, E., Ha¨nel, A., Gossger, K., 2003. Evaluation of the oral immunisation of wild boar against classical swine fever in Baden-Wu¨rttemberg. Berl. Munch. Tierarztl. Wochenschr. 116, 362–367.
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