Research in Veterinary Science 86 (2009) 388–393

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Research in Veterinary Science

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Evaluation of baits for oral vaccination of European wild boar piglets

Cristina Ballesteros a, Christian Gortázar a, Mario Canales a, Joaquín Vicente a, Angelo Lasagna a,José A. Gamarra a, Ricardo Carrasco-García a, José de la Fuente a,b,*

a Instituto de Investigación en Recursos Cinegéticos (IREC) (CSIC-UCLM-JCCM), Ronda de Toledo s/n, 13005 Ciudad Real, Spainb Department of Veterinary Pathobiology, Center of Veterinary Health Sciences, Oklahoma State University, Stillwater, OK 74078, USA

a r t i c l e i n f o

Article history:Accepted 2 September 2008

Keywords:Oral vaccineWildlifeBaitWild boarPigTuberculosisPigletBm95MSP1a

0034-5288/$ - see front matter � 2008 Elsevier Ltd. Adoi:10.1016/j.rvsc.2008.09.003

* Corresponding author. Address: Instituto de Invgéticos (IREC) (CSIC-UCLM-JCCM), Ronda de Toledo sTel.: +34 926 295450.

E-mail addresses: jose_delafuente@yahoo.com, jo(José de la Fuente).

a b s t r a c t

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

� 2008 Elsevier Ltd. All rights reserved.

1. Introduction

European wild boar (Sus scrofa) is an important reservoir hostfor pathogens that affect humans and domestic animals. Pathogenssuch as Mycobacterium bovis (bovine tuberculosis), classical swinefever virus (hog cholera) and porcine herpesvirus (pseudorabies)are maintained in nature due to transmission among wild boarand pigs and the eradication of these diseases may require thedevelopment of control strategies that reduce pathogen transmis-sion among wild boar and also to other species (Brauer et al., 2006;Ballesteros et al., 2007; Cross et al., 2007; Martín-Hernando et al.,2007; Naranjo et al., 2007).

Disease control through vaccination of wildlife has advantagesover other approaches such as population control and is far moreacceptable to the public (Kaden et al., 2005; Cross et al., 2007).However, the effective vaccination of wildlife species such as wildboar requires the development of baits that are effective for theoral delivery of vaccine preparations, stable and preferably, host-specific (Brauer et al., 2006; Ballesteros et al., 2007). This taskhas proven particularly difficult for 2–4 month-old wild boar pig-lets, the preferred age for vaccination (Brauer et al., 2006).

ll rights reserved.

estigación en Recursos Cine-/n, 13005 Ciudad Real, Spain.

se_de_la_fuente@okstate.edu

Therefore, the objective of this study was to develop and evalu-ate new baits for the oral delivery of vaccine preparations to 2–4 month-old European wild boar piglets. Bait physical stabilitywas studied at different temperatures and treatments. Recombi-nant Escherichia coli were used to test bacterial viability in the baitsand the antibody response in orally immunized animals. Baitacceptance was evaluated in uptake studies with wild boar pigletsin the field. The results of these experiments support the use ofthese baits for oral delivery of vaccine formulations to wild boarpiglets.

2. Materials and methods

2.1. Baits

Baits were prepared with a matrix containing 44% piglet feed(37% barley, 32% wheat, 10.5% soy flour, 6% peas, 5% wheat bran,4% corn gluten, 2.3% fatty acids, 1.2% di-calcium phosphate, 1.1%calcium carbonate, 0.5% minerals, vitamins and additives (9000 Uvitamin A, 1800 U vitamin D3, 15 mg copper, 0.7% lysine), and0.4% sodium chloride; Piensos Inalsa S.A., Ciudad Real, Spain),22% wheat flour, 16.5% paraffin (51–53 �C melting point; DilaboS.A., Madrid, Spain), 16.5% sacarose, and 1% cinnamon-truffle pow-der attractant (Norel S.A., Madrid, Spain).

To prepare the bait matrix, paraffin was first melted at 52 �C,sacarose was added and then the rest of the ingredients were

C. Ballesteros et al. / Research in Veterinary Science 86 (2009) 388–393 389

added slowly with constant stirring. The attractant was added atthe end when the bait matrix temperature was approx. 45 �C.The bait matrix was introduced into silicon moulds and polypro-pylene or polyethylene 0.2 ml capsules were dipped into the baitmatrix to introduce vaccine formulation. The baits were allowedto cool at 25 �C, removed from the moulds and used immediatelyfor analysis. The baits had a hemispherical shape (ø3.4 � 1.6 cm)(Fig. 1).

2.2. Physical stability test

Four groups of 21 baits each were incubated at different tem-peratures (4 �C, 25 �C, 37 �C and 42 �C). A fifth group was incubatedin water at 25 �C. Samples of three baits each were taken at differ-ent time points (0, 10, 24, 34, 48, 58 and 72 h) and tested for phys-ical stability by determining the height of the bait after putting onthem a pressure of 111 g/cm2 for 10 min. The difference betweenthe initial and final bait height was then calculated and used forstatistical analysis.

2.3. Bacterial viability test

Escherichia coli K-12 strain JM109 (E. coli) transformed with theexpression vector pMBXAF3 (Canales et al., 2008) were used to testbacterial viability in the baits. Recombinant E. coli were inoculatedin Luria–Bertani (LB) broth containing 50 lg/ml ampicillin and0.4% glucose. Cultures were grown at 37 �C to OD600nm = 2.7 ± 0.3.The cells were harvested by centrifugation at 3800g for 10 min at4 �C and washed with PBS. E. coli cells were resuspended in PBSto 5 � 107 cells/ml and 100 ll were used to fill the capsules thatwere dipped into the bait matrix. When the bait matrix solidified,36 baits were divided into three groups of 12 baits each and incu-bated for up to 96 h at 25 �C, 37 �C and 45 �C. Samples of three baitseach were collected at 24, 48, 72 and 96 h and used to recover thecapsules to test bacterial viability. Recovered E. coli cells were seri-ally diluted in PBS (1:10–1:105) and plated on LB/ampicillin agarplates. The experiment was performed with polyethylene andpolypropylene 0.2 ml capsules. The number of colony formingunits (CFU) was determined after each treatment and the resultswere analyzed statistically.

2.4. Bait acceptance test

Bait acceptance was evaluated in two uptake studies with 2–3 month-old wild boar piglets using selective feeders. The feeders

Fig. 1. Baits (ø3.4�1.6 cm) used for th

consisted of a metal-grid cage with an opening to allow access ofpiglets only. The studies were conducted in a hunting estate inthe province of Ciudad Real, Castilla-La Mancha, central Spain(38�550N; 0�360E; 600–850 m above sea level). In this estate, artifi-cial feeders with maize and pellets are used to supplement the feedof free-ranging wild boar piglets.

In the first study, different proportions of the cinnamon-truffleattractant were used in the baits. Groups of 12 baits each were pre-pared containing 1% (group A) and 2% (group B) attractant. GroupsA and B baits were mixed with maize and pellets and laid out at 10AM in two different selective feeders where wild boar piglets nor-mally feed. Bait uptake was evaluated by monitoring the feedingsites with digital game cameras with infrared illumination (LeafRiver Outdoor products, Taylorsville, MS, USA) and revisiting thefeeding site after 24 h to record complete or incomplete baitconsumption.

In the second study, six selective feeders (called A–F) were ran-domly distributed along all the estate. Twenty five baits were dis-tributed at each feeder and mixed with the rest of the feed. Eachbait contained 1% cinnamon–truffle attractant and a 0.2 ml poly-ethylene capsule filled with water. Bait uptake was evaluatedevery 24 h for four days as described above.

2.5. Oral bait vaccination trial

The capacity of oral baits to induce an immune response in wildboar piglets was evaluated using baits containing recombinantE. coli transformed with the expression plasmid pMBXAF3 as amodel. The E. coli transformed with the expression plasmidpMBXAF3 were grown in Luria–Bertani (LB) broth containing50 lg/ml ampicillin and 0.4% glucose at 37 �C to OD600nm = 0.4. Iso-propyl-b-D-thiogalactopyranoside (IPTG) was then added to0.5 mM final concentration, and incubation continued during3.5 h for induction of expression of the recombinant protein. Theserecombinant bacteria produce upon induction a fusion protein(BM95-MSP1a) comprising tick BM95 immunogenic peptidesfused to the Anaplasma marginale MSP1a N-terminal region thatis displayed on the E. coli surface (Canales et al., 2008). The expres-sion of the recombinant protein was corroborated by SDS–PAGEand reached 2.8% of total cell proteins. Cells were harvested as de-scribed above, resuspended in PBS to 5 � 109 cells/ml and 200 llwere used to fill the polyethylene capsules that were dipped intothe bait matrix.

Four 2–3 month-old wild boar piglets were used in the experi-ment. Wild boar were bred in captivity at the Senda Viva Park

e studies described in the paper.

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Fig. 2. Bait physical stability and bacterial viability tests. (A) Physical stability test:four groups of 21 baits each were incubated at different temperatures. A fifth groupwas incubated in water at 25 �C. Samples of three baits each were taken at differenttime points and tested for physical stability by determining the height of the baitafter putting a pressure on them of 111 g/cm2 for 10 min. The difference betweenthe initial and final bait height was then calculated and used for statistical analysis.(B) Bacterial viability test: groups of 12 baits each were incubated at differenttemperatures. Samples of three baits each were collected at different time pointsand used to recover the polyethylene 0.2 ml capsules to test bacterial viability.Recombinant E. coli cells were diluted in PBS and plated on LB/ampicillin agarplates. The number of colony forming units (CFU) was determined and comparedbetween treatments using a factorial ANOVA test which included time andtreatment as explanatory factors and the 2-way interaction followed by post-hocTurkey tests to test differences between treatments (*P < 0.05). Values shown aregeometric means ± SD (N = 3).

390 C. Ballesteros et al. / Research in Veterinary Science 86 (2009) 388–393

(Arguedas, Navarra, Spain), free of tuberculosis and without signsof other diseases. Animals were housed under controlled condi-tions at the Senda Viva Park with the approval and supervision ofpark authorities. Pellets and water were supplied ad libitum. Ani-mals were randomly distributed into two groups using the RandomNumber Generator Pro v1.45 (Segobit Software, http://www.seg-obit.com/rng.htm). Two immunized boars (Nos. 026 and 035) re-ceived six recombinant E. coli-containing baits each. Two controlboars (036 and 095) received six PBS-containing baits each. Baitswere given orally on days one, 21 and 42 of the experiment. Bloodwas collected for serum preparation on days 1 (before oral baitadministration) and 56 (two weeks after last bait administration).Antibodies to BM95-MSP1a fusion protein were determined byELISA and western-blot.

Fecal samples were collected from immunized boars on day 70(four weeks after last bait administration) and cultured in MacCon-key agar plates and LB agar plates containing 50 lg/ml ampicillin(LBA plates). Forty eight bacterial colonies derived from LBA platesof each immunized boar were grown as described above and usedto check the presence of the pMBXAF3 plasmid by PCR using vec-tor-specific oligonucleotide primers (N26: 50-CAT CAT AAC GGTTCT GGC AAA TAT TC-30 and C24: 50-CTG TAT CAG GCT GAA AATCTT CTC-30; Sigma–Aldrich, St. Louis, MO, USA).

2.6. SDS–polyacrylamide gel electrophoresis (PAGE) and western-blotanalysis

About 10 lg of total protein from induced recombinant E. coli(pMBXAF3) were loaded into a 12% PAGEgelTM SDS cassette gel(PAGE-gel, San Diego, CA, USA) and either stained with CoomassieBrilliant Blue or transferred to a nitrocellulose membrane for wes-tern-blot analysis. For western-blot analysis, proteins in the gelwere transferred to a nitrocellulose membrane (Schleicher & Schu-ell, PROTRAN BA85, Dassel, Germany) and probed with wild boarsera diluted 1:10 in 3% BSA in tris-buffered saline (TBS) as de-scribed previously (Canales et al., 2008). Serum from an adult (>2years-old) free-ranging wild boar (No. 001) with detectable anti-E. coli antibodies was also included in the experiment to examinethe cross-reactivity of naturally acquired anti-E. coli antibodies.The membrane was finally incubated with a rabbit anti-pig horse-radish peroxidase (HRP) conjugate (Sigma–Aldrich) diluted1:10,000 in TBS. The membrane was washed 10 times with TBSand finally developed with TMB stabilized substrate for HRP (Pro-mega) for 5 min.

2.7. Analysis of antibody titers by ELISA

Antibody responses against the BM95-MSP1a fusion proteinwere evaluated by indirect ELISA in immunized and control wildboar piglets. Partially purified recombinant protein (0.1 lg perwell) was used to coat ELISA plates over night at 4 �C. Sera wereserially diluted from 1:10 to 1:320 in PBS/0.05% Tween 20 (PBST)and 5% skim milk. The plates were incubated with the diluted serafor 1 h at 25 �C and then incubated with 1:500 rabbit anti-pig HPRconjugate (Sigma) for 1 h at 25 �C. The color reaction was devel-oped with SIGMAFAST OPD (o-Phenylenediamine dihydrochloride)(Sigma) and the OD450nm was determined. Plates were washedwith PBST between incubations. Antibody titers were consideredpositive when yielded an OD value at least twice as high as thenegative control serum and were expressed as the geometric mean(mean ± SD) at the 1:10 serum dilution (N = 2).

2.8. Statistical analysis

For the analysis of physical stability test results, the differencebetween the initial and final bait height was used as response var-

iable to test the effect of time and treatment over the studied per-iod. A general linear model (GLM, SPSS 14.0, Chicago. IL, USA) wasused with a factorial ANOVA structure which included time (0, 10,24, 34, 48, 58 and 72 h) and treatment (4 �C, 25 �C, 37 �C, 42 �C andwater) as explanatory factors and the 2-way interaction. A similarmodel was built for E. coli viability after treatment, for which log10transformed CFU was used as dependent variable, and time (24, 48,72 and 96 h) and temperature (25 �C, 37 �C and 45 �C) as explana-tory factors and the 2-way interaction. Post-hoc Turkey tests wereused to test differences between categories. Statistical uncertaintywas expressed through 95% confidence intervals of the standarderror (or standard deviation). The ELISA antibody titers againstthe BM95-MSP1a fusion protein were compared between immu-nized and control wild boar piglets by Student’s t-test (P < 0.05).

3. Results

Baits were designed and prepared with a hemispherical shape(ø3.4 � 1.6 cm) using a matrix composed of wild boar feed, wheat

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flour, paraffin, sacarose, and cinnamon-truffle powder attractant(Fig. 1). The bait matrix should guarantee that the bait is stable un-der field conditions so the vaccine could reach target species.Physical stability tests demonstrated that baits were stable for atleast three days at 42 �C (Fig. 2A). However, water did affect baitphysical stability after two days (Fig. 2A).

The bait matrix composition and preparation should also pro-tect the vaccine formulation to keep its efficacy. The paraffinselected for bait matrix composition had a melting point of 51–53 �C, a temperature that did not affect E. coli viability in polyeth-ylene capsules after bait preparation (98.7 ± 10.6% viabilityexpressed as the number of CFU recovered after approximately30 min when the bait reached room temperature). Although someloss of viability was observed after bait incubation at 37 �C and45 �C, the E. coli viability was not significantly affected after baitincubation at 25 �C and 37 �C for 96 h (Fig. 2B). The E. coli viabilitywas affected only after 72 h incubation at 45 �C (Fig. 2B). The poly-propylene capsules were less effective in protecting E. coli viability(data not shown). Polypropylene capsules showed a decrease of12.0 ± 2.7% in E. coli viability after bait preparation and a significantdecrease in E. coli viability after bait incubation at 37 �C for 96 hand at 45 �C for 48 h.

Field uptake studies demonstrated that baits were accepted by2–3 month-old wild boar piglets, the preferred age for vaccination.In the first study, the baits in both groups (containing 1% and 2%cinnamon-truffle attractant) were completely consumed after24 h by wild boar piglets, as expected mostly during the eveninghours (Fig. 3). Two replicate experiments were conducted withsimilar results.

Fig. 3. Nocturnal photograph of 2–3 month-old wild boar piglets feeding

Fig. 4. Polyethylene 0.2 ml capsules chewed and spit o

In the second study, six feeders (named A–F) received 25 baitseach and were examined each morning for four days to recordthe number of baits consumed and intact or chewed capsulesfound. In the feeders A–C, all the 25 baits placed on each feederwere consumed during the first night. In feeder D, 4 baits werefound at the first revision but they were all consumed during thesecond night. In feeder E, 19 baits were consumed during the firstnight and the six remaining baits were consumed during the sec-ond night. In feeder F animals needed four nights to consume allthe baits, probably due to the fact that this feeder was less visitedby wild boars piglets than the other feeders. Out of the 150 poly-ethylene capsules inserted into the baits, only 52 were recovered.In all but two occasions, recovered capsules were chewed andempty (Fig. 4). Therefore, it is very likely that most of the capsules(65%) were eaten and swallowed by wild boars piglets and only 1%of the capsules remained intact after bait ingestion.

Wild boar piglets were orally immunized with baits containingrecombinant E. coli that expressed the recombinant membrane-displayed BM95-MSP1a fusion protein after induction as modelto evaluate the capacity of oral baits to induce an immune re-sponse in wild boar piglets (Fig. 5A). The wild boar accepted thebaits and the recombinant E. coli-containing polyethylene capsuleswere either swallowed or chewed and spit out empty by the ani-mals after bait ingestion. Immunized animals had low but signifi-cant (P = 0.04) IgG antibody titers against the recombinant fusionprotein (OD values at 1:10 serum dilution of 14 ± 7 and 0 ± 6 forimmunized and control animals, respectively) and a band corre-sponding to the BM95-MSP1a fusion protein was detected bywestern-blot in wild boar orally immunized with recombinant

on artificial feeders containing baits mixed with maize and pellets.

ut empty after bait ingestion by wild boar piglets.

Fig. 5. Immune response of wild boar piglets vaccinated with the recombinantE. coli expressing the BM95-MSP1a fusion protein. (A) Recombinant E. coli(pMBXAF3) were induced with IPTG for 3.5 h for the expression of recombinantBM95-MSP1a fusion protein (arrow). Ten micrograms of total protein were loadedper well in a 10% SDS–PAGE. The gel was stained with Coomassie Brilliant Blue.Uninduced E. coli (pMBXAF3) was included as controls. (B) For western-blotanalysis, induced E. coli (pMBXAF3) proteins were transferred to a nitrocellulosemembrane, probed with wild boar antisera (026 and 035, immunized; 036, 095 and001, controls) and the signal developed with a rabbit anti-pig HRP conjugate. Theposition of the recombinant BM95-MSP1a fusion protein is indicated with arrows.Color Burst electrophoresis markers (Sigma) were used as molecular weight (MW)markers in the electrophoresis.

392 C. Ballesteros et al. / Research in Veterinary Science 86 (2009) 388–393

E. coli-containing baits but not in controls (Fig. 5B). A positive sig-nal was not detected with preimmune sera and the serum from theadult free-ranging wild boar, thus ruling out cross-reactivity ofnaturally acquired anti-E. coli antibodies with the recombinantprotein (Fig. 5B and data not shown).

Ampicillin-resistant E. coli were recovered from the feces of vac-cinated wild boar in a number similar to bacterial colonies growingin MacConkey agar plates. However, 36% of the colonies growingon LBA plates were shown to be recombinant E. coli-containingthe pMBXAF3 plasmid.

4. Discussion

The control and eradication of diseases such as bovine tubercu-losis and hog cholera may require the immunization of wild boarpiglets in some regions (Kaden et al., 2000; Kaden and Lange,2001; Kaden et al., 2005; Ballesteros et al., 2007; Martín-Hernandoet al., 2007; Naranjo et al., 2007). The oral immunization of wildboar piglets at 2–3 months of life is difficult due to physiologicalproblems associated with the immunological gap (Kaden et al.,2002, 2003, 2005) and limited bait uptake (Brauer et al., 2006) ofthese animals. Therefore, the objective of our study was to developbaits for the oral immunization of 2–3 month-old European wildboar piglets, the preferred age for vaccination.

The results of physical stability and bacterial viability tests sug-gested that the baits described herein are stable and suitable forfield application during the summer months, when young wildboar population increases in regions such as south-central Spainwhere mean monthly maximum temperatures reach 34.5 �C in Au-gust, and rainfall is minimal with less than 30 mm in the periodJuly–September (Vicente et al., 2004, 2005). Polyethylene capsulesresulted in higher bacterial viability after bait preparation and afterincubation at 37 �C and 45 �C. Lower bacterial viability in the poly-propylene capsules probably resulted from higher heat exchangein these capsules due to thinner walls when compared to the poly-ethylene capsules.

With the ingredients used for bait matrix composition, it waspredicted that the baits would be highly palatable to wild boar pig-

lets and would stimulate chewing to rupture the capsule inside itand deliver vaccine to the oropharyngeal lymphoid tissues of theanimals. The baits were completely consumed by 2–3 month-oldwild boar piglets. After bait ingestion by wild boar piglets, theE. coli-containing polyethylene capsules were either swallowedor chewed and spit out empty by the animals, thus likely releasingits content inside the animal’s oral cavity.

Placing the baits inside selective feeders for wild boar pigletsensured that only this age group was in contact with the oral baitformulation. However, selective feeders for wild boar piglets arenot always present in hunting estates and other sites where wildboars live. Therefore, experiments are underway in our laboratoryusing mobile feeders to test the consumption of baits by wild boarpiglets in places where selective feeders are absent.

The final proof of vaccine oral delivery to wild boar was ob-tained in preliminary experiments with BM95-MSP1a-expressingE. coli-containing baits. Polyethylene capsules were selected forthese experiments because these capsules preserved E. coli viabil-ity better than the polypropylene capsules. Wild boars were orallyimmunized with a high antigen content formulation. Wild boar re-ceived a total of 18 baits in three administrations of 6 baits each.Each bait contained 109 E. coli cells, equivalent to approx. 4 mg re-combinant protein (considering an expression level of 2.8% of totalcell proteins and 0.15 g total protein content for 109 E. coli cells).Therefore, each immunized wild boar received a total of approx.24 mg recombinant protein on each oral immunization (72 mgafter three immunizations). However, wild boar that received baitscontaining BM95-MSP1a-expressing E. coli developed low antibodytiters specific against the recombinant protein, as determined twoweeks after the last bait administration. In this study, the inductionof Th1-type cytokines, responsible for killing intracellular patho-gens (Berger, 2000) was not evaluated.

Recombinant E. coli cells containing the pMBXAF3 plasmid wererecovered from the feces of vaccinated wild boar four weeks afterthe last bait administration, thus confirming that vaccine composi-tion was released and reached the wild boar gastrointestinal track.Ampicillin-resistant E. coli that did not contain the pMBXAF3 plas-mid and were therefore not related to the vaccine formulationwere isolated from wild boar feces. E. coli are an important com-mensal and pathogen that inhabits the gastrointestinal tracts ofhumans and domestic and wild animals (Hartl and Dykhuizen,1984), and it is regarded as an important source of antimicrobialresistance determinants for other human and animal pathogens(Neu, 1992).

The oral immunization with recombinant antigens has beendemonstrated in different animal species but very few oral vac-cines have been licensed (Silin et al., 2007). However, most oralvaccine formulations currently under development for wild boarand other wildlife species are based on live infective organismsand the induction of Th1 response, which should produce a morerobust protective immunity after oral vaccination (Blanton et al.,2007; Collins et al., 2007; Cross et al., 2007; Cross and Aldwell,2007; Koenig et al., 2007; Shakya et al., 2007).

Brauer et al. (2006) reported that oral vaccination of young wildboar during the first three months of life is impossible. This prob-lem was associated with the fact that wild boar piglets are sucklingduring the first three months of life, a time when they come in con-tact with solid food (Brauer et al., 2006). However, under the con-ditions found in Spanish hunting estates, wild boar piglets alreadyconsume solid food after two months of age (Fig. 3 and unpub-lished observations). It is possible that under our conditions wildboar piglets become adapted to solid food earlier due to the factthat artificial feeders are made accessible to this age group, with-out competition with older animals. The adaptation to the solidfood may stimulate bait uptake by 2–3 month-old wild boar pig-lets. This hypothesis is confirmed by studies in which higher bait

C. Ballesteros et al. / Research in Veterinary Science 86 (2009) 388–393 393

uptake was obtained in wild boar and feral pigs fed with placebosbefore the onset of the trial (Hohne and Stone, 1989; McIlroy et al.,1989; Saunders et al., 1990; Kaden et al., 2000). Additionally, short-er suckling times may overcome the immunological gap reportedby Kaden et al. (2002, 2003, 2005) in wild boar up to the age of3–4 months, thus making possible active immunization in youngeranimals as shown in our studies.

The host-specificity of the baits described herein was not ad-dressed in these studies. However, the use of artificial feeders forbait administration to wild boar piglets may prevent or reduce baituptake by other species (unpublished observations).

In summary, our results indicate that the baits described hereincould be used for the oral immunization of 2–4 month-old wildboar piglets, during summers with relatively high temperaturesand at least in places where artificial feeders are used to supple-ment the feed of wild boar piglets. However, the experiments re-ported herein were done using recombinant E. coli as a model toevaluate the capacity of baits to induce an immune response in wildboar piglets. Trials involving vaccination with relevant antigens andchallenge for the control of wild boar infections are needed to fullyaddress the efficacy of oral baits developed in this work.

Acknowledgements

This work was supported by Grants from Instituto Nacional deInvestigación y Tecnología Agraria y Alimentaria (INIA) (projectFAU 2006-00017-C03-01), Consejería de Educación y Ciencia, Juntade Comunidades de Castilla-La Mancha (JCCM) (Project PAI 06-0046-5285), the Grupo Santander and the Fundación MarcelinoBotín, Spain. Cristina Ballesteros is a recipient of a JCCM fellowship.

José M. Pérez de la Lastra and Rafael Reyes (IREC) are acknowl-edged for technical assistance. This work is a contribution to theagreement between Yolanda Fierro and the University of Castilla-La Mancha.

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