Classical swine fever virus Strain 'C'. How long is it detectable after oral vaccination?

To determine the persistence period of C-strain vaccine virus in immunized animals, domestic pigs and wild boars were vaccinated orally and killed on different days post vaccinationem (dpv). Tissue samples were taken at necropsy from both species for detection of C-strain virus. From domestic pigs nasal swabs and faeces were also collected. During the investigation period (2-12 dpv) vaccine virus could never be detected in nasal secretions and in faeces of vaccinated domestic pigs. In contrast, C-strain virus was found in organs until day 8 pv in domestic pigs and until day 9 pv in wild boars. Whereas in domestic pigs virus was detected in tonsils, Ln. mandibularis or in spleen, in wild boar it only was found in tonsils. We conclude that C-strain vaccine virus is not detectable in wild boars longer than 10-12 days after intake of the vaccine baits.     

Oral immunisation of swine with a classical swine fever vaccine (Chinese strain) and transmission studies in rabbits and sheep

Seven experiments including a total of 47 pigs, 11 wild boars, 26 rabbits, 10 hares and 16 sheep were carried out to assess the efficacy, safety and transmission of the Chinese vaccine strain of the classical swine fever virus (CSFV) administrated by the oral route. Within 3 weeks after oral vaccination, a clear seroconversion occurred in the pigs. Six weeks after vaccination, vaccinated pigs were fully protected against a virulent challenge. The C-strain was not isolated from tonsils, spleen, lymph nodes, thymus, saliva, urine and faeces of pigs within 4 days after oral vaccination. In one experiment, susceptible pigs were placed in direct contact with vaccinated pigs. None of these contact-exposed pigs became serologically positive for CSFV antibodies. It is concluded that the C-strain induces protection in pigs when administrated by the oral route and is not shed by vaccinated pigs. Serum anti-CSFV antibodies developed in seven out of eight wild boars vaccinated by the oral route. No vaccine virus was detected in the spleen and tonsils of these animals. The results in wild boar were in accordance with those obtained in domestic pigs. Sheep did not show any clinical signs after oral vaccination while rabbits had moderate hyperthermia and growth retardation. No clinical response to oral immunisation in hares was detected. At the end of the experiment, no sheep had detectable serum antibodies against CSFV, whereas a few vaccinated rabbits and hares became seropositive. None of the contact-exposed rabbits and hares seroconverted. These data indicate that the C-strain is safe for sheep and as expected, moderately or not pathogenic for rabbits and hares. These efficacy and safety studies on oral vaccination with the C-strain under experimental conditions provide essential information for further studies in wild boars under experimental and field conditions, including assays with baits to control a CSF epidemic.     

Genetic differentiation of infected from vaccinated animals after implementation of an emergency vaccination strategy against classical swine fever in wild boar

Oral emergency vaccination against classical swine fever is a powerful tool to control disease outbreaks among European wild boar and thus to safeguard domestic pigs in affected regions. In the past, when virus detection was mainly done using virus isolation in cell culture or antigen enzyme-linked immunosorbent assays, modified live vaccine strains like C-strain "Riems", were barely detectable after oral vaccination campaigns. Nowadays, the use of highly sensitive molecular techniques has given rise to an increase in vaccine virus detections. This was also the case during the 2009 outbreak among German wild boar and the subsequent vaccination campaigns. To guarantee a rapid differentiation of truly infected from C-strain vaccinated animals, a combination of differentiating multiplex rRT-PCR assays with partial sequencing was implemented. Here, we report on the rational and use of this approach and the lessons learned during execution. It was shown that positive results in the recently developed vaccine strain (genotype) specific rRT-PCR assay can be taken as almost evidentiary whereas negative results should be confirmed by partial sequencing. Thus, combination of multiplex rRT-PCR assays as a first line differentiation with partial sequencing can be recommended for a genetic DIVA strategy in areas with oral vaccination against classical swine fever in wild boars.     

Classical Swine Fever Virus Strain ‘C’. How Long is it Detectable After Oral Vaccination?

To determine the persistence period of C‐strain vaccine virus in immunized animals, domestic pigs and wild boars were vaccinated orally and killed on different days post vaccinationem (dpv). Tissue samples were taken at necropsy from both species for detection of C‐strain virus. From domestic pigs nasal swabs and faeces were also collected. During the investigation period (2–12 dpv) vaccine virus could never be detected in nasal secretions and in faeces of vaccinated domestic pigs. In contrast, C‐strain virus was found in organs until day 8 pv in domestic pigs and until day 9 pv in wild boars. Whereas in domestic pigs virus was detected in tonsils, Ln. mandibularis or in spleen, in wild boar it only was found in tonsils. We conclude that C‐strain vaccine virus is not detectable in wild boars longer than 10–12 days after intake of the vaccine baits.     

Vaccination of Tunisian dogs with the lyophilised SAG2 oral rabies vaccine incorporated into the DBL2 dog bait

The protective effect of the lyophilised SAG2 oral vaccine bait DBL2, already demonstrated on laboratory dogs, needed to be verified on common Tunisian dogs. Seven Tunisian dogs consumed totally or partially one DBL2 bait containing 10(8.3) TCID50 of the highly attenuated rabies vaccine strain, SAG2. Five of the seven vaccinated animals survived a challenge administered 33 days later with a Tunisian canine street rabies virus to which five of the six controls that were not vaccinated and had no specific antibodies succumbed. The partial or total consumption of a single DBL2 bait thus conferred a protective immune response similar to that observed in laboratory dogs to dogs of poor health status. The sero-antibody response was, however, weak: only two vaccinated dogs exhibited a significant neutralising antibody response after vaccination and before the challenge, and four after the challenge.     

Evaluation of baits for oral vaccination of European wild boar piglets

The objective of this study was to develop and evaluate new baits for the oral delivery of vaccine preparations to 2–4 month-old wild boar piglets. Baits were prepared using a matrix composed of wild boar feed, wheat flour, paraffin, sacarose and cinnamon-truffle powder attractant with polyethylene capsules dipped into the matrix to introduce vaccine formulation. Physical stability studies demonstrated that baits were stable for at least three days at temperatures as high as 42 °C. Recombinant Escherichia coli expressing the membrane-displayed BM95-MSP1a fusion protein were used to test bacterial viability in the baits and the antibody response in orally immunized wild boar. The E. coli viability was not significantly affected after bait incubation at 25 and 37 °C for 96 h. Bait acceptance studies using artificial feeders in the field showed that baits were accepted by 2–3 month-old animals, the preferred age for vaccination. Orally immunized wild boar piglets excreted recombinant E. coli in the feces and developed antibody titers to recombinant BM95-MSP1a protein, thus confirming that vaccine composition was released and reached the wild boar gastrointestinal track. The results of these experiments support the use of these baits for oral delivery of vaccine formulations to 2–4 month-old wild boar piglets.     

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.     

Vaccination of feral pigs (Sus scrofa) using iophenoxic acid as a simulated vaccine

OBJECTIVES: To develop an encapsulation method for delivery of vaccines to feral pigs, and quantify the effect of iophenoxic acid on captive feral pig blood iodine concentrations to assist in investigation of factors affecting vaccine uptake. DESIGN AND METHODS: Feral pigs were administered iophenoxic acid by oral gavage, and consumption was assessed for different encapsulation methods in baits. Blood iodine concentrations were monitored for eight days after consumption. The relationship between dose rate, time since dosing and blood iodine concentration was assessed for gavaged and baited captive feral pigs. Wild feral pigs were baited with PIGOUT baits containing 20 mg of encapsulated iophenoxic acid to simulate a vaccination program. Using knowledge from the pen studies, bait uptake and factors affecting bait uptake were investigated. RESULTS: Bait-delivered iophenoxic acid led to variable and inconsistent changes in blood iodine concentrations, in contrast to pigs receiving iophenoxic acid by gavage. This precluded accurate assessment of the quantity consumed, but still allowed a conservative determination of bait uptake. Iophenoxic acid in smaller capsules was consumed readily. Increasing baiting intensity appeared to increase bait uptake by wild feral pigs, and pigs of varying sexes, ages and weights appeared equally likely to consume baits. CONCLUSIONS: Encapsulated liquids can be delivered to feral pigs within baits, should the need to vaccinate feral pigs for fertility or disease management arise. High baiting intensities may be required.     

Long-term studies on maternal immunity for Aujeszky's disease and classical swine fever in wild boar piglets

The aim of the studies was to fathom the duration and the role of maternal immunity for Aujeszky's disease (AD) and classical swine fever (CSF) in wild boar offspring. In one experiment, two wild boar sows were infected with a low pathogenic pseudorabies virus (PRV) in 1999. A total of 51 offspring was born between 1999 and 2002 and was monitored for PRV maternal antibodies. In a second experiment, the maternal immunity for CSF was analysed. Therefore, a sow was orally vaccinated against CSF using vaccine baits containing the live-attenuated C-strain vaccine. The vaccination took place in January 1999. The sow gave birth to four piglets in 2001 and to two piglets in 2002. With respect to maternal immunity for AD, some piglets reacted positive in the ELISA up to 27-week post-partum while in the neutralization test antibodies were detected up to 15-week post-partum. The calculated half-life of neutralizing antibodies was 21 days. Regarding CSF, the neutralization titres of maternal antibodies dropped continuously reaching values of < or =10 ND50 20-week post-partum. After the 12th week post-partum, most of the sera reacted negative in the ELISA. However, after the third month, low levels of neutralization titres were still detectable. The results are discussed with respect to the epidemiology and control of both diseases in wild boar populations.     

Development of maternal antibodies after oral vaccination of young female wild boar against classical swine fever

An experimental study was performed to investigate the development of maternal antibodies after oral immunisation of young female wild boar against classical swine fever (CSF) using C-strain vaccine. Our results demonstrated that maternal antibodies do not persist in the offspring for more than 3 months. Based on the neutralising serum antibody titres, we assume that piglets of wild sows vaccinated orally twice or immunised once a long time before conception have protective antibodies for approximately 2 months. Furthermore, it seems that the level and the duration of maternal antibodies in the offspring are depend on the age of the female animals at the moment of vaccination as demonstrated in our experiment. The recent vaccination procedure consists of three double vaccinations in spring, summer and autumn. Especially vaccinations in summer and autumn could be crucial for transfer of high maternal antibody titres to the offspring.     

Safety and immunogenicity of ERA strain of rabies virus propagated in a BHK-21 cell line.

The ERA strain of rabies virus was propagated in a baby hamster kidney cell line (BHK-21/C13). The viral titer was 10(1.8) tissue culture infective doses (TCID) higher than that of commercial ERA vaccine. The ERA/BHK-21 vaccine in baits retained titers of 10(6.3) to 10(6.4), TCID when subjected to daily temperature fluctuations from 9 degrees C to 24 degrees C for 21 days. This titer, according to a dose response in laboratory foxes, was still capable of immunizing up to 100% of foxes consuming a bait. The ERA/BHK-21 vaccine, when presented in baits, produced antibodies in 80 to 100% of dogs consuming more than one bait. Duration of immunity in foxes, from feeding the ERA strain rabies virus in baits, as determined by resistance to challenge with virulent virus, was at least 48 months. The vaccine strain retained some pathogenicity for nontarget species. In tests carried out on foxes, raccoons, dogs, cats and cattle, the vaccine did not cause vaccine-induced rabies. One of 14 skunks which consumed four baits developed vaccine-induced rabies, but virus could not be isolated from the salivary glands of this animal. The vaccine, when presented in baits, caused vaccine-induced rabies in 37% of laboratory mice, 3.4% of Microtus and 2.6% of Peromyscus species. Rabies virus could not be isolated from the salivary glands of rodents with vaccine-induced rabies. It was concluded that ERA virus propagated in BHK-21/C13 cells and incorporated in an acceptable bait produced a high titer, stable, immunogenic and safe vaccine for foxes.     

Studies on efficacy and stability of a vaccine bait containing ERA strain of rabies virus propagated in a BHK-21 cell line.

In a dose response study in foxes, the median protective dose of ERA BHK21 vaccine in a blister pack bait was 10(6.0) tissue culture infective doses (TCID)/mL, while artificially aged baits with titers of 10(6.3) TCID/mL induced seroconversion in 78% of foxes. There was no significant difference in the development of antibodies in foxes receiving 1, 2 or 3 mL volumes of vaccine in the bait. When baits were exposed to the elements and fed to foxes over a 21 day period, 85% of the animals seroconverted. Age, sex and the way in which the vaccine container was contacted did not appear to be factors in the responses of these animals. Juvenile foxes, approximately six months of age, were marked more readily with the tetracycline bait marker than older animals. Approximately 25% of foxes did not appear to respond well to vaccination and the titer of the vaccine was a critical factor in producing seroconversion in these animals.     

Acceptance and palatability for domestic and wildlife hosts of baits designed to deliver a tuberculosis vaccine to wild boar piglets

Tuberculosis (TB) caused by Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex, is an important health problem worldwide. The control of TB through vaccination of wildlife reservoirs may potentially have advantages over other management strategies. The most practical approach to deliver vaccines to wildlife is using oral baits that are stable under field conditions and effective in reaching the target species. Baits were developed in our laboratory to deliver oral vaccines to wild boar piglets. However, these baits were well accepted by other wild species. Therefore, bait consumption by different M. bovis hosts was evaluated herein. The results showed that the baits were well accepted by cattle, feral pigs, and adult red deer whereas small mammals like badgers and possums showed varying bait acceptance. Bait acceptance by different species has the advantage of targeting more than one wildlife reservoir when they coexist in the same area and need to be vaccinated for TB control. However, bait delivery methods such as the use of selective feeders to target the desired species should be developed to avoid bait consumption by other species.     

Efficacy of E2-sub-unit marker and C-strain vaccines in reducing horizontal transmission of classical swine fever virus in weaner pigs

At present, two types of vaccines against classical swine fever (CSF) virus are commercially available: E2 sub-unit marker vaccines and the conventional attenuated live C-strain vaccines. To evaluate the reduction of the horizontal virus transmission, three comparable experiments were carried out in which groups of weaner pigs (vaccinated with a marker vaccine or a C-strain vaccine) were challenged with CSF virus at 0, 7, and 14 days post-vaccination (dpv). Virus transmission was prevented totally when the challenge occurred at 14 dpv with an E2-marker vaccine (0/12 contact pigs positive in virus isolation (VI); R = 0 (0; 1.5)). At 7 dpv, transmission was reduced slightly (5/12 contact pigs positive in VI; R = 1.0 (0.3; 3.0)), whereas at 0 dpv, vaccination had no effect on transmission (10/12 contact pigs positive in VI; R = 2.9 (1.5; 10.8)). In the C-strain-vaccinated pigs, no virus transmission was detected even when the challenge was performed at the same day as the vaccination (0/12 contact pigs positive in VI; R = 0 (0; 1.5)).     

Oral immunisation of wild boar against classical swine fever: evaluation of the first field study in Germany

The effectiveness of oral immunisation of wild boar against classical swine fever (CSF) was studied in a field trial in Lower Saxony for two years, from 1993 to 1995. This field study was performed in an area of ca. 270 km(2)50% of young boars did not feed on vaccine baits nor become immunised. Therefore, an intensive hunting of this age group is a necessary adjunct to the use of oral vaccination. After the third immunisation period, no virus was detected in the areas where oral immunisation took place.     

Experimental infection of wild boar and domestic pigs with a foot and mouth disease virus strain detected in the southeast of Bulgaria in December of 2010

Foot and mouth disease (FMD) was detected in a wild boar in Southeastern Bulgaria in December 2010. The occurrence and spread of the disease in wild cloven-hoofed animals may pose an unexpected and significant threat to FMD virus (FMDV)-free areas within and outside the European Union. So far, only one well documented experimental infection with FMD in wild boar has been published. In order to obtain more epidemiologically relevant data regarding the disease in wild boar we conducted an experiment with the 2010 Bulgarian FMDV type O isolate. Two young wild boar were challenged while two domestic pigs and two additional wild boar served as contact controls. While the domestic pigs developed severe clinical signs of FMD, the wild boar showed relatively mild course of the disease. Viremia started in contact wild boar 2 days post exposure (DPE) and lasted until 6 DPE. The virus shedding lasted until 9 DPE. On 27 DPE, when the animals were slaughtered, viral RNA was detected in lymphoid tissues and oropharyngeal fluid but no virus could be isolated. Commercial ELISAs and virus neutralisation tests detected antibodies against FMDV on 8 or 6 DPE, respectively. The data of the present study will help to understand FMD in wild boar populations and can be used in models to evaluate the potential role of wild boar in FMD epidemiology.     

Vaccinology of classical swine fever: from lab to field

There are two types of classical swine fever vaccines available: the classical live and the recently developed E2 subunit vaccines. The live Chinese strain vaccine is the most widely used. After a single vaccination, it confers solid immunity within a few days that appears to persist lifelong. The E2 subunit vaccine induces immunity from approximately 10-14 days after a single vaccination. The immunity may persist for more than a year, but is then not complete. The Chinese strain vaccine may establish a strong herd immunity 1-2 weeks earlier than the E2 vaccine. The ability of the Chinese vaccine strain to prevent congenital infection has not been reported, but the E2 subunit vaccine does not induce complete protection against congenital infection. Immunological mechanisms that underlie the protective immunity are still to be elucidated. Both types of vaccine are considered to be safe. A great advantage of the E2 subunit vaccine is that it allows differentiation of infected pigs from vaccinated pigs and is referred to as a DIVA vaccine. However, the companion diagnostic E(rns) ELISA to actually make that differentiation should be improved. Many approaches to develop novel vaccines have been described, but none of these is likely to result in a new DIVA vaccine reaching the market in the next 5-10 years. Countries where classical swine fever is endemic can best control the infection by systematic vaccination campaigns, accompanied by the normal diagnostic procedures and control measures. Oral vaccination of wild boar may contribute to lowering the incidence of classical swine fever, and consequently diminishing the threat of virus introduction into domestic pigs. Free countries should not vaccinate and should be highly alert to rapidly diagnose any new outbreak. Once a new introduction of classical swine fever virus in dense pig areas has been confirmed, an emergency vaccination programme should be immediately instituted, for maximum benefit. The question is whether the time is ripe to seriously consider global eradication of classical swine fever virus.     

Evaluation of the oral immunisation of wild boar against classical swine fever in Baden-Württemberg

The oral immunisation of wild boar against classical swine fever (CSF) in Baden-Württemberg is described and evaluated. The bait vaccine based on the CSF virus (CSFV) strain "C" proved to be safe in wild boar of all age classes. The modified immunisation procedure consisting of three double vaccinations per year was very effective. CSFV was not detected beyond the second immunisation campaign. The average rate of seropositive wild boar diagnosed over all immunisation periods was 49.2%. The seroprevalence rate increased significantly during the first year of immunisation and reached its maximum after the third vaccination period with 72% antibody positive animals. The higher percentage of seropositive young boars in this field trial compared to the seroprevalence rates in this age class in other field trials in Germany may be attributed to the new vaccination scheme. Factors that may be responsible for the decreased herd immunity after the fourth or sixth immunisation period are discussed.     

Long‐Term Studies on Maternal Immunity for Aujeszky's Disease and Classical Swine Fever in Wild Boar Piglets

The aim of the studies was to fathom the duration and the role of maternal immunity for Aujeszky's disease (AD) and classical swine fever (CSF) in wild boar offspring. In one experiment, two wild boar sows were infected with a low pathogenic pseudorabies virus (PRV) in 1999. A total of 51 offspring was born between 1999 and 2002 and was monitored for PRV maternal antibodies. In a second experiment, the maternal immunity for CSF was analysed. Therefore, a sow was orally vaccinated against CSF using vaccine baits containing the live‐attenuated C‐strain vaccine. The vaccination took place in January 1999. The sow gave birth to four piglets in 2001 and to two piglets in 2002. With respect to maternal immunity for AD, some piglets reacted positive in the ELISA up to 27‐week post‐partum while in the neutralization test antibodies were detected up to 15‐week post‐partum. The calculated half‐life of neutralizing antibodies was 21 days. Regarding CSF, the neutralization titres of maternal antibodies dropped continuously reaching values of ≤10 ND₅₀ 20‐week post‐partum. After the 12th week post‐partum, most of the sera reacted negative in the ELISA. However, after the third month, low levels of neutralization titres were still detectable. The results are discussed with respect to the epidemiology and control of both diseases in wild boar populations.     

Oral immunisation of wild boar against classical swine fever: concluding analysis of the recent field trials in Germany

The recent oral immunisation trials in wild boar against classical swine fever (CSF) in Germany are described and evaluated in summary. After the first field study in Lower Saxony from 1993-1995 further immunisation trials started in Mecklenburg-Western Pomerania, Brandenburg, Lower Saxony, Baden-Württemberg and Saxony-Anhalt. The immunisation strategies and the size of the vaccination zones were different in the individual federal states. In principle, the bait vaccine based on the CSF virus strain "C" were laid out by hand. Later also the aerial distribution was carried out in selected areas of Mecklenburg-Western Pomerania. The application of baits by plane was introduced at the beginning of the immunisation measures in Saxony-Anhalt apart from the manual distribution. Up to now, the field trials show that the oral immunisation can be an additional tool for CSF control by increasing of herd immunity and reduction of the CSFV prevalence. However, the immunisation was not sufficient enough for young boars in the most field studies. Based on the evaluation of the immunisation experiments an improved immunisation procedure is recommended.