Bait vaccine

Abstract

The present disclosure provides a bait vaccine formulation for pigs, the formulation comprising an antigen; the present invention relates to a bait formulation comprising an antigen, optionally an adjuvant and a matrix, where the antigen is inside a container, where the container is entirely within the matrix, where the matrix is not coated with a protective film, where the bait formulation is temperature and / or humidity stable and / or suitable for deployment in the air. Methods of using these bait vaccine formulations are also provided.

Description

[0001] Names of the parties to the joint research agreement

[0002] The claimed invention is the result of activities carried out within the scope of a joint research agreement between Zoetis LLC and IREC (UCLM-CSIC). Technical Field

[0003] The present invention generally belongs to the field of bait vaccines for protecting wild boars. Background Art

[0004] Disease control in wild animals is a significant challenge (Delahay et al., 2009), and oral vaccination is one of the few tools available for combating the most serious infectious diseases shared by wild animals (Gortázar et al., 2015). It is used to reduce the number of susceptible animals below the threshold for maintaining infection (Rupprecht et al., 2004; Blancou et al., 2009), or to mitigate the severity of the disease, thereby reducing the spread of infection (Garrido et al., 2011).

[0005] The Eurasian wild boar (Sus scrofa) is a natural reservoir for several pathogens shared with humans and farm animals, and thus effective disease control is necessary to mitigate the consequences of the presence of shared infections (Ballesteros et al., 2007). Oral vaccination has great potential to control infections in wild reservoirs and prevent outbreaks in other species (Müller et al., 2012). In Europe, and in the specific case of wild boars, the first field tests were carried out in the 1990s to control classical swine fever (CSF), and later the oral vaccination of wild boars against CSF was extended to different European countries (authorizing regulation UE 2020 / 689). For this process, different factors must be considered, such as the early delivery of vaccination, the landscape structure and the spatial distribution of food sources, as well as the professional support of cooperation with hunters and wildlife and forestry agencies (Rossi et al., 2015).

[0006] Vaccines can seek contact with the oropharyngeal mucosa (Ballesteros et al., 2007) or seek to cross the gastric barrier without inactivating the antigen by acids and enzymes to reach the intestine. The latter can be achieved in two ways, one is through lipid composition baits (Aldwell et al., 2003), because fats are attacked by bile acids in the small intestine, and the other is through capsules or other protective containers dissolved in the intestine (Mahato et al., 2003). African swine fever vaccines are most likely to target the oral mucosa (Sang et al., 2020).

[0007] Effective oral vaccination of wild animals requires the development of baits that are effective, stable, and preferably host-specific for orally administered vaccines (Brauer et al., 2006; Ballesteros et al., 2007). This task is particularly difficult for piglets aged 2 to 4 months, which is the ideal age for CSF vaccination (Brauer et al., 2006). Precedent baits include the bait (Kaden et al., 2000) used for classical swine fever vaccination, the Australian wild boar bait (Cowled et al., 2006) used to deliver poison to feral pigs in Australia and the United States, and the IREC bait (Ballesteros et al., 2009) used for oral vaccination against animal tuberculosis in Spain.

[0008] However, these baits have certain disadvantages. The bait has a low melting point (30 °C), and its shape and texture can make the bait difficult to handle and result in vaccine loss (Rossi et al., 2015). Additionally, this bait is too large for wild boars under 4 months of age to consume (Faust et al., 2007). The bait requires high temperatures during the extrusion process of its production (Beltrán-Beck et al., 2013).

[0009] The IREC bait proposed by Ballesteros et al. (2009) was designed to contain 0.2 ml polyethylene capsules to introduce the vaccine formulation. These baits use paraffin (melting point 51 °C to 53 °C) as an adhesive and were found to be suitable for field applications in summer when the temperature is relatively high (up to 44 °C, Beltrán-Beck et al., 2014). However, these baits have limited moisture resistance (Ballesteros et al., 2009) and are not species-specific (Ballesteros et al., 2011; Beltrán-Beck et al., 2014).

[0010] In addition, in previous wild boar vaccination attempts, the low uptake of baits by piglets under 6 months of age has been a problem because of the low vaccination rate at this age (Brauer et al., 2006; Rossi et al., 2011; Sage et al., 2011; Calenge and Rossi, 2014). Field trials using the IREC bait achieved up to 92% bait uptake by piglets, mainly by using selective feeders targeted at piglets (Ballesteros et al., 2011; Díez-Delgado et al., 2019).

[0011] An effective and specific baiting strategy for the species to be vaccinated is one of the most challenging tasks because there are multiple non-target species, such as birds or predators, which may interfere with bait uptake. To prevent birds from consuming the bait, which is also an issue when using rodenticides for pest control, the possibility of using a certain coloring agent as a deterrent has been studied, and the effectiveness of using blue and green colors to reduce bird consumption of the bait has been verified (Cowan et al., 2017).

[0012] Baits have been applied through many baiting strategies, such as aerial application by plane or helicopter (Kaden et al., 2002), burying them to specifically target wild boars and protect live vaccines from high temperatures (Kaden et al., 2002), through selective piglet feeders (Ballesteros et al., 2009), or placing the bait under heavy stones that wild boars can lift (Díez-Delgado et al., 2019). Aerial baiting is particularly suitable for large-area interventions (Siers et al., 2017). However, IREC-type baits have never been dropped from an aircraft, and there is a lack of assessment of bait resistance.

[0013] Therefore, there is a need in the art for improved baits for vaccination of suids (wild boars and pigs). Summary of the Invention

[0014] The present disclosure addresses these and other needs in the art by providing, in a first aspect, a bait vaccine formulation for pigs, the formulation comprising:

[0015] a. an antigen,

[0016] b. an optional adjuvant,

[0017] c. a matrix, wherein the antigen is inside a container, wherein the container is completely within the matrix, wherein the matrix is not coated with a protective film, and wherein the bait formulation is

[0018] i) temperature-stable and / or

[0019] ii) humidity-stable and / or

[0020] iii) suitable for aerial deployment.

[0021] In some embodiments, the matrix comprises corn flour, starter feed, sugar, and an adhesive, wherein the starter feed comprises from about 30% w / w to about 42% w / w barley, from about 30% w / w to about 35% w / w wheat, from about 9% w / w to about 11% w / w soybean meal, from about 3% w / w to about 10% peas, from about 2% w / w to about 8% wheat bran, from about 1% w / w to about 7% w / w corn bran, and from about 1.5% to about 3% w / w fatty acids. Preferably, the starter feed comprises about 37% w / w barley, about 32% w / w wheat, about 10.5% w / w soybean meal, about 6% w / w peas, about 5% w / w wheat bran, about 4% w / w corn bran, about 2.3% w / w fatty acids, and further comprises about 1.2% w / w dicalcium phosphate, about 1.1% w / w calcium carbonate, about 0.5% w / w minerals, vitamin A, vitamin D3, copper, lysine, and sodium chloride. More preferably, the corn flour is present in the matrix at about 14% w / w to about 22% w / w, the starter feed is present in the matrix at about 35% w / w to about 45% w / w, the sugar is present in the matrix at about 12% w / w to about 18% w / w, and the adhesive is present in the matrix at about 24% w / w to about 28% w / w. In other preferred embodiments, wherein the corn flour is present in the matrix at about 17% w / w, the starter feed is present in the matrix at 40 to 42% w / w, the sugar is present in the matrix at 14 to 16% w / w, and the adhesive is present in the matrix at 24 to 26% w / w.

[0022] In some embodiments applicable to any of the above compositions, the adhesive is honey or cane sugar. In further additional or alternative embodiments also applicable to any of the above compositions, the sugar is sucrose.

[0023] Preferably, in the bait of any of the above embodiments, the antigen is within a container, and wherein the container is entirely within the matrix.

[0024] In some embodiments, the bait is generally oblong in shape, wherein the longest dimension is at least 1.8 times the second longest dimension, and wherein the longest dimension is between about 2.5 cm and about 6 cm. In a more preferred embodiment, the bait vaccine formulation has a generally hemispherical shape, wherein the longer dimension is about 2.5 to about 3.6 cm, and the shorter dimension is about 1 to about 1.7 cm.

[0025] In some embodiments of the present invention applicable to any of the above bait formulations, the formulation is weight-stable at 25°C to 42°C.

[0026] In further additional or alternative embodiments of the present invention applicable to any of the above bait formulations, the formulation is humidity-stable.

[0027] In a further additional or alternative embodiment of the invention applicable to any of the above bait formulations, the formulation is suitable for aerial delivery.

[0028] In certain embodiments applicable to any of the formulations described herein, the antigen is selected from the group consisting of antigens that provide protection against infection, and the infection is selected from the group consisting of: African swine fever virus, classical swine fever virus, Orf virus, and Mycobacterium tuberculosis complex and any combination thereof. In a more preferred embodiment, the antigen is an ASF antigen.

[0029] In certain embodiments applicable to any of the formulations described herein, the bait vaccine formulation is black, green, or blue.

[0030] In a second aspect, the present disclosure provides a method of eliciting a protective immune response against a pathogen in a population of wild or feral pigs in a habitat, the method comprising placing a bait formulation according to any embodiment of the first aspect of the invention in the habitat, wherein the antigen elicits a protective response against the infection. In certain embodiments of this second aspect of the invention, the bait formulation is delivered by aerial delivery.

[0031] In a third aspect, the present disclosure provides the use of a bait formulation according to any embodiment of the first aspect for eliciting a protective immune response against a pathogen in wild or feral pigs. In certain embodiments of this third aspect, the bait formulation is delivered by aerial delivery. Detailed Description

[0032] When used in conjunction with a measurable numerical variable, the terms "about" or "approximately" refer to the indicated value of the variable and all values of the variable within the experimental error of the indicated value (e.g., within the 95% confidence interval of the mean) or within 10% of the indicated value, whichever is greater.

[0033] The term "antigen" refers to any substance that is recognized by an animal's immune system and elicits an immune response. The term encompasses killed, inactivated, attenuated, or modified live bacteria, viruses, or parasites. The term "antigen" also includes polynucleotides, polypeptides, recombinant proteins, synthetic peptides, protein extracts, cells (including tumor cells), tissues, polysaccharides, or lipids or fragments thereof, alone or in any combination. The term "antigen" also encompasses antibodies, such as anti-idiotypic antibodies or fragments thereof, and synthetic peptide mimotopes that can mimic an antigen or antigenic determinant (epitope).

[0034] The term "humidity-stable" refers to the weight change of the bait preparation after immersing no more than 7% of the bait preparation in water for 72 hours at 25°C to 42°C. Thus, if the weight change is no more than 5% compared to the weight before the test under the above conditions, the preparation is humidity-stable. In a preferred embodiment, the weight change of the humidity-stable preparation is no more than 4%, or no more than 3%, or no more than 2%, or no more than 1%.

[0035] The term "oblong shape" refers to a shape having a longer and a shorter dimension, and wherein the cross-section is substantially the same as or smaller than the shorter dimension. Preferably, the longest dimension is at least 1.8 times the second longest dimension. In a more preferred embodiment, the longer dimension is 1.8 to 2.5 times the second longer dimension, including but not limited to about 1.9 times, 2 times, 2.1 times, 2.2 times, 2.3 times, and 2.4 times. Preferably, the edges of the oblong shape are rounded for easier consumption. Without limitation, the oblong shape can be in the form of a prism, a cylinder, or an oval shape. In certain preferred embodiments, the oblong shape is hemispherical, sized about 2.5 to about 4 cm by about 1 to about 2 cm.

[0036] The phrase "suitable for aerial delivery" refers to a displacement of less than 8 mm as measured in the examples provided.

[0037] The term "pathogen" is used together with an antigen. Briefly, the antigen is capable of eliciting a protective immune response against the corresponding pathogen.

[0038] The term "protective immune response" refers to a reduction in the intensity or duration of at least one clinical symptom of a given pathogen infection. The clinical symptoms vary depending on the nature of the pathogen and can include but are not limited to fever, diarrhea, lung lesions, nasal discharge, oral discharge, fecal discharge, and other symptoms. Preferably, the protective immune response prevents the vaccinated animal from being infected with the pathogen. The protective immune response can be measured directly or inferred through immunological endpoints, including markers of the cell-mediated immune system and protective antibody titers.

[0039] The term "vaccine" refers to a composition containing an antigen, wherein the composition elicits a protective immune response against the corresponding pathogen.

[0040] In a broad aspect, the present invention provides a bait vaccine preparation for pigs, the preparation comprising: an antigen; an optional adjuvant; and a matrix, wherein the antigen is inside a container, wherein the container is completely within the matrix, wherein the matrix is not coated with a protective film, and wherein the bait preparation is temperature-stable and / or humidity-stable and / or suitable for aerial delivery.

[0041] The matrix of the bait comprises corn flour, piglet feed, sugar and a binder. Preferably, the binder is honey or cane molasses. More preferably, the binder is honey. Generally, the corn flour is present in the matrix at about 14% w / w to about 22% w / w, the piglet feed is present in the matrix at about 35% w / w to about 45% w / w, the sugar is present in the matrix at about 12% w / w to about 18% w / w, and the binder is present in the matrix at about 24% w / w to about 28% w / w.

[0042] In other embodiments, the corn flour is present in the matrix at about 14% w / w to 21% w / w, the piglet feed is present in the matrix at about 35% w / w to 43% w / w, the sugar is present in the matrix at about 12% w / w to about 18% w / w, and the binder (e.g., honey) is present in the matrix at about 24% w / w to about 28% w / w.

[0043] In a further preferred embodiment, wherein the corn flour is present in the matrix at about 17% w / w, the piglet feed is present in the matrix at 40 to 42% w / w, the sugar is present in the matrix at 14 to 16% w / w, and honey is present in the matrix at 24 to 26% w / w.

[0044] In any of the above matrix compositions, the piglet feed comprises about 30% w / w to about 42% w / w of barley, about 30% w / w to about 35% w / w of wheat, about 9% w / w to about 11% w / w of soybean meal, about 3% w / w to about 10% of peas, about 2% w / w to about 8% of wheat bran, about 1% w / w to about 7% w / w of corn bran and about 1.5% to about 3% w / w of fatty acids. It should be understood that in the discussion of "w / w" percentages applied to piglet feed, the percentages are given relative to the total composition of the piglet feed rather than the total composition of the bait matrix. The piglet feed may also contain minerals such as copper, vitamins (e.g., vitamin A and vitamin D3), amino acids (such as polylysine) and sodium chloride.

[0045] In a more specific embodiment that is also applicable to any of the above matrix compositions, the piglet feed comprises about 37% w / w of barley, about 32% w / w of wheat, about 10.5% w / w of soybean meal, about 6% w / w of peas, about 5% w / w of wheat bran, about 4% w / w of corn bran, about 2.3% w / w of fatty acids, and further comprises about 1.2% w / w of dicalcium phosphate, about 1.1% w / w of calcium carbonate, about 0.5% w / w of minerals, vitamin A, vitamin D3, copper, lysine and sodium chloride (about 0.4% w / w).

[0046] Natural beeswax has been used as a superhydrophobic coating (Li et al., 2018). However, the inventors have observed that wild boars tend to damage the coated bait and consume the exposed substrate. This is undesirable as it may lead to the loss of Eppendorf tubes and thus vaccination failure. Therefore, preferably, the bait disclosed herein does not contain any coating.

[0047] In a particularly preferred embodiment, in the substrate, corn flour is present at about 17% w / w of the substrate, the sugar is present at 14 to 16% w / w of the substrate, honey is present at 24.5 to 25.5% w / w of the substrate, and the piglet feed is present at 40 to 42% w / w of the substrate, wherein the piglet feed comprises about 37% w / w of barley, about 32% w / w of wheat, about 10.5% w / w of soybean meal, about 6% w / w of peas, about 5% w / w of wheat bran, about 4% w / w of corn bran, about 2.3% w / w of fatty acids, and further comprises about 1.2% w / w of dicalcium phosphate, about 1.1% w / w of calcium carbonate, about 0.5% w / w of minerals, vitamin A, vitamin D3, copper, lysine and about 0.4% of sodium chloride.

[0048] The bait of the present invention is generally oblong in shape, including cylindrical, semi-cylindrical, hemispherical, oval, semi-elliptical, prismatic, etc. Preferably, the angles of these shapes are rounded for easier consumption. Preferably, the bait should be generally hemispherical. The size of the bait is limited by the size of the animal. Currently preferred is that the long dimension should be about 2.5 to about 6 cm, and the longer dimension should be 1.8 to 2.5 times the shorter dimension. In the most preferred embodiment, the bait is hemispherical, wherein the longer dimension is about 2.9 to about 3.8 cm, and the shorter dimension is about 1 to about 1.8 cm. In the most preferred embodiment, the bait is hemispherical, wherein the longer dimension is about 2.9 to about 3.5 cm, and the shorter dimension is about 1 to about 1.5 cm.

[0049] The bait of the present invention generally has at least one of the following properties - they are heat-resistant, they are moisture-resistant, and / or they are suitable for aerial delivery. In a more preferred embodiment, at least two of these properties are present, and in an even more preferred embodiment, all three properties are present. The temperature-stable bait of the present invention does not change weight after 72 hours in the temperature range of 25°C to 42°C, and particularly in the temperature range of 37°C to 42°C. The humidity-stable bait of the present invention does not change weight after being immersed in about 7% water for up to 72 hours. In a particularly preferred embodiment, exposure to temperatures from 4°C to 42°C up to 48°C and humidity (submersion, as described in the examples) does not affect the impact resistance of the bait of the present invention.

[0050] The matrix of the bait contains a container that houses the antigen and optionally an adjuvant. Preferably, the container is entirely within the matrix. The volume of the container should be sufficient to accommodate the antigen and optionally the adjuvant. In practice, the volume of the container can vary between about 1 ml and about 0.1 ml, including but not limited to about 0.2 ml, about 0.3 ml, about 0.4 ml, about 0.5 ml, about 0.6 ml, about 0.7 ml, about 0.8 ml, about 0.9 ml. In certain embodiments applicable to any of the baits described herein, the volume of the container is about 0.2 ml.

[0051] The container can be biodegradable or non - biodegradable. Suitable non - limiting materials for the container include but are not limited to cellulose, PLA / PGA polymers, and plastics. In certain embodiments, the container is a vial.

[0052] A variety of antigens are suitable for administration via the bait. The most suitable antigens can elicit a protective immune response against pathogens that affect wild boar and feral pig populations and can be transmitted to farmed pigs. More specifically, the pathogens include but are not limited to African swine fever virus, classical swine fever virus, pseudorabies virus. In other embodiments, the pathogens can be of bacterial or protozoal origin, including but not limited to members of the Mycobacterium tuberculosis complex and other mycobacteria, Lawsonia intracellularis, Giardia, Cryptosporidium spp, and other pathogens.

[0053] Both monovalent vaccines (antigens against one pathogen) and multivalent vaccines (antigens against multiple pathogens) can be present in the container. The antigen can be in the form of inactivated pathogens (e.g., inactivated bacteria, inactivated viruses, bacterins), modified live pathogens, subunits, and DNA vaccines. Combinations of these antigens are also possible.

[0054] The container can also contain an optional adjuvant. A variety of adjuvants are known in the art, and the choice of adjuvant will depend on the nature of the antigen. Suitable adjuvants include but are not limited to aluminum compounds, mycobacterial cell walls, saponins (including their complexes with sterols and / or phospholipids, such as for example )), lipopolysaccharides, poly I:C, CpG - containing immunostimulatory oligonucleotides, glycolipids, liposomes. Different combinations of these adjuvants are also possible.

[0055] In addition to the antigen and optionally the adjuvant, various modifications are possible for the bait vaccine formulation suitable for administration with the baits described herein. For example, the contents of the container can include mucoadhesives, carriers, antibiotics, antiparasitics, other drugs, buffers, preservatives, etc.

[0056] The bait of the present invention can be delivered by a variety of methods. For example, in habitats where wild boar diets are supplemented with feed such as corn or piglet feed, the bait can be left together with the corn or piglet feed. In other embodiments, the bait can be left in places where pigs are known to frequent, such as near farmland or pig tracks. Other methods, such as area delivery, delivery in selective piglet rearing cages, and delivery under heavy stones, are also possible.

[0057] In Europe, aerial bait delivery has proven successful in fox rabies control (Müller et al., 2012). Aircraft delivery of bait has also been used to control certain species with toxic bait, such as possums (Trichosurus vulpecula) in New Zealand (Morgan, 2010), feral cats (Felis silvestris) and red foxes in Australia (Moseby et al., 2011), or invasive brown tree snakes (Boiga irregularis) in Guam (Goetz et al., 2021). However, no prior wild boar bait has been tested for suitability for aerial delivery. As described in the examples, the inventors used a compression test to simulate area delivery to measure the displacement that might be caused by the interaction of the bait with the ground. When the displacement was greater than 8 mm, it was considered that the impact plane collided with the container containing the antigen and optionally the adjuvant. Such a collision could cause rupture. Considering this limitation, it was observed that the new bait formulation resisted impacts from a maximum height of 500 meters. Thus, in different embodiments, the bait formulation can resist dispersion at about 100 to 500 meters above the ground, or about 200 to 500 meters above the ground, or about 300 to 500 meters above the ground, or about 400 to 500 meters above the ground, or about 100 to about 400 meters above the ground, or about 200 to about 300 meters above the ground.

[0058] The following examples are presented as illustrative embodiments and should not be regarded as limiting the scope of the present invention. Many changes, variations, modifications, and other uses and applications of the present invention will be apparent to those skilled in the art.

[0059] Examples

[0060] Example 1: Composition and Properties of the Bait

[0061] Materials and Methods

[0062] Use of Animals in Bait Palatability Tests

[0063] This study was conducted using non-invasive photo-capture techniques and did not involve the capture, handling, or sampling of any animals. The protocol was designed by specifically trained and certified scientists in accordance with EC Directive 86 / 609 / EEC and was approved by the Animal Experimentation Committee of the University of Castilla-La Mancha and the Regional Ethics Committee (PR-2022-01-01).

[0064] Bait production and composition

[0065] The starting point was the IREC bait, which was designed to contain a 0.2 ml polyethylene capsule for introducing the vaccine preparation. The bait was prepared with a matrix containing 44% piglet feed, 22% wheat flour, 16.5% paraffin wax (Dilabo SA, Madrid, Spain), 16.5% sucrose, and 1% cinnamon and truffle attractant powder (Norel SA, Madrid, Spain) (Ballesteros et al., 2009). Different components and protective films were tested to improve the mechanical properties of the bait and its tolerance to environmental temperature and humidity. Finally, the improved bait matrix composition included 41% piglet feed (Piensos Inalsa, Ciudad Real, Spain), 17% corn flour, 15% sucrose, and 25% honey. Honey served as an adhesive instead of using paraffin wax. Both the IREC and the new bait contained a 200 μl Eppendorf tube (VWR, Pennsylvania, USA) at the center of the bait matrix to carry the target vaccine or substance.

[0066] Palatability tests

[0067] Palatability tests were conducted in the field to rule out some particularities in the production of these baits. The experiments examined protective coatings (waxed bait vs. ordinary bait), different shapes (spherical vs. hemispherical), composition (the IREC preparation proposed by Ballesteros et al. (2009) vs. other formulations), various odors: vanilla-cinnamon-truffle (Norel S.A., Madrid, Spain), anise (PME Cake, Riverside Business Park, UK), almond (PME Cake, Riverside Business Park, UK), cadaverine (≥97.0% GC, Sigma-Aldrich, Darmstadt, Germany); and colorants (black, green, and blue; Wilton Brands LLC, Illinois, USA) to arrive at the most suitable composition (Table 1). These tests were carried out in hunting areas where wild boars were completely or partially free, with the diet of wild boars supplemented with corn. The detection time (Td, the time for wild boars to detect each bait group) and consumption time (Tc, the time for wild boars to completely consume each bait group) were calculated.

[0068] Field tests were conducted using only camera traps (10 tests, including field experiments where birds were offered bait). In these final field tests (bird field tests), five baits of four different colors were compared. The baits were distributed one in front of the other such that five hidden cameras (set for 48 hours) would separately capture the bait type preferences of wild boars and non-target species. In each test, the cameras recorded the number of visits by each species and the proportion of bait consumed. Visits were continuously filmed for 48 hours, capturing everything until the bait would be completely depleted. A 10-minute interval was considered to define a new visit.

[0069] Bait physical stability

[0070] Humidity

[0071] To quantify the effect of humidity on the physical stability of the bait, three different sets of baits, consisting of a total of 14 baits, were separately exposed to 3 conditions: a saturated medium (with a maximum relative humidity percentage of 100% at 25 ℃ °C) (H1), a saturated medium but with a water depth of 1 mm (the medium in which the bait remained semi-submerged, 100% at 25 °C) (H2), and another medium in which the bait was completely submerged (15 °C) (H3). Samples were taken at 0, 10, 24, 34, 48, 58, and 72 hours. For comparison, three sets of IREC baits (8 baits per set) were tested under the same conditions. Samples were taken at 0, 24, 48, and 72 hours.

[0072] At the designated sampling times, the baits were weighed and dropped from a height of 5 meters onto a concrete surface. This process was repeated up to 15 times for each bait, and the number of impacts on the ground that the bait could withstand without losing the Eppendorf tube was recorded.

[0073] Temperature

[0074] Four different sets, each consisting of 14 baits, were made and separately exposed to 4 °C, 25 °C, 37 °C, and 42 °C. Samples were taken at 0, 10, 24, 34, 48, 58, and 72 hours. For comparison, four sets, each consisting of 8 IREC baits, were exposed to the same temperatures as above, and samples were collected at 0, 24, 48, and 72 hours.

[0075] Simulation of aerial bait delivery

[0076] The simulation was run using SolidWorks CAD software (SolidWorks Corp., Dassault Systèmes, Suresnes, France). The compression tests were performed using an electromechanical universal testing machine from Instron Corporation (Instron 5696, Illinois ToolWorks Inc., Glenview, IL, USA), which was equipped with a 1 kN load cell and a compression platen. The load was applied at a constant displacement rate of 5 mm / min. The specimens used for this test were cylindrical, made of the same material as the bait, with a height L = 60 mm and a diameter D = 44 mm. During the test, a 3D digital image correlation (DIC) system commercialized by Correlated Solutions Inc. (Irmo, SC, USA) was used for the optical measurement of material displacement and strain. The DIC parameters used for these tests were: subset size = 85 pixels; step size = 11 pixels. Once the displacement field was obtained by correlating the specimen images acquired every 4 seconds during the test, the strain was derived due to subsequent processing of the data using VIC 3D software (Correlated Solutions Inc., Irmo, SC, USA). The stress was calculated as follows:

[0077]

[0078] where F is the applied load, and A = π(D / 2) 2 is the initial cross-sectional area. Once the strain was obtained using the DIC technique, the Poisson's ratio (v) was obtained as the ratio of the strain ε yy to ε xx which are the strains averaged in the transverse (y-direction) and longitudinal (x-direction) directions in the target area:

[0079]

[0080] The elastic modulus (Young's modulus) was the slope of the stress-strain curve in the linear region calculated by linear regression. Finally, the yield strength was obtained as the stress level at the onset of nonlinearity in the stress-strain curve.

[0081] Statistical analysis

[0082] For the statistical analysis of the palatability tests, the Mann-Whitney U test and the Kruskal-Wallis H test were selected to examine the waxes used, as recorded in R (R Core Team, 2018) to allow for more than two groups to be considered. When doing so, the results obtained in the physical stability tests of humidity and temperature showed that both the variable "weight gain" and the variable "number of impacts" were considered dependent variables. Similarly, the effects of composition and treatment were recorded over a 72-hour range. A GLM with a full factorial model was proposed using IBM SPSS Statistics 24 (Chicago, Illinois, USA).

[0083] Results

[0084] Palatability tests

[0085] Table 1 presents the results of seven palatability trials conducted on captive wild boars. Although none of the trials resulted in statistically significant differences between the prototypes, some relevant insights were obtained. First and foremost, regardless of the bait matrix tested, the average times for bait detection and bait consumption were nearly the same for IREC compared to the new formulation, indicating that the new formulation does not reduce the bait uptake rate. The bait could be detected simultaneously (as expected) regardless of its shape, but the time for wild boars to consume spherical baits doubled as spherical baits tend to roll away. Additionally, the presence / absence of the wax coating and the taste and color of the bait were not related to bait detection and consumption by wild boars. Cadaverine taste was not tested as it was difficult to introduce into the bait matrix.

[0086] Table 1 summarizes the palatability trials on captive wild boars. The results of seven trials are presented, testing the effects of the presence / absence of the wax coating, spherical versus hemispherical bait shapes, different tastes (compared to the plain bait), and black coloring (compared to no colorant), showing the number of baits, number of replicates, average detection time, and consumption time (in minutes) and their standard errors for each test group.

[0087] Table 1. Effects of Bait Color, Odor, Shape, Matrix Composition on Bait Consumption

[0088]

[0089] Field trials of bait preference

[0090] The camera traps were placed for 48 hours, and the visits of different animal species and the percentage of bait consumption for each species were recorded (Table 2). A total of 96 visits to the bait were recorded, of which 49% were by corvids and 19% were by the target species, wild boar. Regarding bait consumption, wild boars consumed 94% of the bait they approached, while foxes, dogs, and birds consumed 38% - 42%. Table 2 - Species (in %) visiting the bait and bait consumption rate (in %) for each species during the field trial with the new bait formulation. The trial consisted of 10 bait sites with 20 baits per site.

[0091]

[0092]

[0093] To deter birds, different dyes were tested to record the effect of bait color on corvid preference for bait. The results are summarized in Table 3. In four trials, a preference for colorless bait was observed.

[0094] Table 3 - Birds and their bait preferences as evaluated in five field trials comparing baits with different colors.

[0095]

[0096] Except for birds, no animals showed a preference for the different colors used.

[0097] Physical stability

[0098] The physical stability tests regarding humidity and temperature compared the performance of the new bait formulation with that of the IREC bait.

[0099] Humidity

[0100] Baits completely submerged in water (extreme humidity, H3) dissolved and lost their shape after a maximum of 10 hours, regardless of the bait type. For baits placed in a saturated humidity environment (H1) and a saturated and semi-submerged environment (H2), the results of weight gain and resistance to dropping from a 5 m height are shown in Table 4. For H1, it was found that both bait types absorbed little moisture and remained almost stable, while for H2, the IREC bait was severely affected by moisture (Table 4; p > 0.05). Regarding shock resistance, the new bait formulation was superior to the IREC bait. The IREC bait first increased its resistance when it was slightly wetted, but in a semi-submerged environment where it absorbed more humidity, it quickly lost its shock resistance (Table 4; p = 0.036).

[0101] Table 4: Comparison of the effect of humidity on bait weight and shock resistance between the new bait formulation and the IREC bait.

[0102] Bait t ΔP Average number of impacts Humidity New bait formulation 0 0 15 H1 New bait formulation 10 0.682 15 H1 New bait formulation 24 0.7485 15 H1 New bait formulation 34 1.0445 15 H1 New bait formulation 48 0.7745 15 H1 New bait formulation 58 1.02 15 H1 New bait formulation 72 1.4105 15 H1 IREC bait 0 0 6 H1 IREC bait 24 1.128 10.5 H1 IREC bait 48 1.0385 14.5 H1 IREC bait 72 1.3825 12.5 H1 New bait formulation 0 0 15 H2 New bait formulation 10 0.6545 15 H2 New bait formulation 24 1.533 15 H2 New bait formulation 34 1.4615 15 H2 New bait formulation 48 1.5265 15 H2 New bait formulation 58 2.5395 15 H2 New bait formulation 72 2.326 15 H2 IREC bait 0 0 4 H2 IREC bait 24 7.844 1.5 H2 IREC bait 48 12.4235 1.5 H2 IREC bait 72 15.422 1 H2

[0103] Temperature

[0104] As in the previous tests, both bait types were subjected to different temperatures. The weight and number of impacts resisted by the baits were recorded (Table 5). The new formulation bait remained almost stable in terms of temperature, while the IREC bait was prone to weight loss, and there was a significant interaction between bait type and time (p = 0.001). Regarding impact resistance, the new formulation bait was more resistant than the IREC bait (Table 5; p > 0.05).

[0105] Table 5: Comparison of the effects of temperature on bait weight and impact resistance between the new bait formulation and the IREC bait.

[0106]

[0107]

[0108] Aerial bait delivery simulation

[0109] Eight specimens were tested by compression testing and applying the DIC technique, from which five representative test outputs were obtained (Table 6).

[0110] Table 6: Results of the elastic modulus (Young's modulus) and yield strength of the tests conducted.

[0111]

[0112]

[0113] Note that the overall longitudinal strain ε = ΔL / L of the specimen is calculated as the displacement ΔL of the actuator divided by the initial length L of the cylinder. The representation of the stress-strain response using the global strain obtained from the displacement of the actuator allows for a qualitative visualization of the full response of the material during the test. At the same time, DIC strain measurements are used, where the stress-strain evolution is represented until the visual macroscopic damage of the specimen allows for the extraction of a credible strain field. The Poisson's ratio (v) in the linear region is close to 0.5 (0.499). Using this information, the impact simulation was analyzed using SolidWorks. Finally, the average value of the maximum deformation generated in the five representative samples and at each simulated height was recorded.

[0114] Table 7: Average value and standard deviation of the maximum deformation generated in five representative samples for each height.

[0115] H(m) Average maximum deformation SD 200 3,7064 0,5917 500 7,1452 1,1695 750 9,6836 1,4551 1000 11,618 1,8218

[0116] In summary, these data indicate that the lures disclosed herein are more temperature and humidity stable than the IREC lures, are suitable for aerial delivery, and allow for more species-specific consumption without sacrificing palatability.

[0117] All publications (both patent publications and non-patent publications) cited in this specification indicate the level of skill in the art to which the present invention pertains. All of these publications are hereby incorporated by reference in their entirety to the same extent as if each individual publication were specifically and individually indicated to be incorporated by reference.

[0118] Although the present invention has been described herein with reference to specific embodiments, it should be understood that these embodiments merely illustrate the principles and applications of the invention. Accordingly, it is to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be designed without departing from the spirit and scope of the invention as defined by the following claims.

Claims

1. A bait vaccine preparation for pigs, the preparation comprising: a. An antigen; b. An optional adjuvant, c. A matrix, wherein the antigen is inside a container, wherein the container is completely within the matrix, wherein the matrix is not coated with a protective film, and wherein the bait preparation is i) Temperature-stable and / or ii) Humidity-stable and / or iii) Suitable for aerial deployment.

2. The bait preparation according to claim 1, wherein the matrix comprises corn flour, piglet feed, sugar, and an adhesive, and wherein the piglet feed comprises from about 30% w / w to about 42% w / w of barley, from about 30% w / w to about 35% w / w of wheat, from about 9% w / w to about 11% w / w of soybean meal, from about 3% w / w to about 10% of peas, from about 2% w / w to about 8% of wheat bran, from about 1% w / w to about 7% w / w of corn bran, and from about 1.5% to about 3% w / w of fatty acids.

3. The bait preparation according to claim 2, wherein the piglet feed comprises about 37% w / w of barley, about 32% w / w of wheat, about 10.5% w / w of soybean meal, about 6% w / w of peas, about 5% w / w of wheat bran, about 4% w / w of corn bran, about 2.3% w / w of fatty acids, and further comprises about 1.2% w / w of dicalcium phosphate, about 1.1% w / w of calcium carbonate, about 0.5% w / w of minerals, vitamin A, vitamin D3, copper, lysine, and sodium chloride.

4. The bait preparation according to claim 2 or 3, wherein the corn flour is present in the matrix at about 14% w / w to about 22% w / w, the piglet feed is present in the matrix at about 35% w / w to about 45% w / w, the sugar is present in the matrix at about 12% w / w to about 18% w / w, and the adhesive is present in the matrix at about 24% w / w to about 28% w / w.

5. The bait preparation according to any one of claims 2 to 4, wherein the corn flour is present in the matrix at about 17% w / w, the piglet feed is present in the matrix at 40 to 42% w / w, the sugar is present in the matrix at 14 to 16% w / w, and the adhesive is present in the matrix at 24 to 26% w / w.

6. The bait preparation according to any one of claims 2 to 5, wherein the adhesive is honey or cane sugar.

7. The bait preparation according to any one of claims 2 to 6, wherein the sugar is sucrose.

8. The bait preparation according to any one of claims 1 to 7, wherein the antigen is inside a container, and wherein the container is completely within the matrix.

9. The bait preparation according to any one of claims 1 to 8, wherein the bait preparation is generally oblong in shape, wherein the longest dimension is at least 1.8 times the second-longest dimension, and wherein the longest dimension is between about 2.5 cm and about 6 cm.

10. The bait preparation according to any one of claims 1 to 9, wherein the preparation is weight-stable at 25°C to 42°C.

11. The bait preparation according to any one of claims 1 to 10, wherein the preparation is humidity-stable.

12. The bait preparation according to any one of claims 1 to 10, wherein the preparation is suitable for aerial deployment.

13. The bait preparation according to any one of claims 1 to 12, wherein the bait vaccine preparation has a generally hemispherical shape, wherein the longer dimension is about 2.5 to about 3.6 cm, and the shorter dimension is about 1 to about 1.7 cm.

14. The bait preparation according to any one of claims 1 to 13, wherein the antigen is an ASF antigen.

15. The bait preparation according to any one of claims 1 to 14, wherein the antigen is selected from the group consisting of antigens that protect against infection, the infection being selected from the group consisting of: African swine fever virus, classical swine fever virus, Orzykowski's disease virus, and Mycobacterium tuberculosis complex and any combination thereof.

16. The bait preparation according to any one of claims 1 to 15, wherein the bait vaccine preparation is black, green, or blue.

17. A method of eliciting a protective immune response against a pathogen in a population of wild or feral pigs in a habitat, the method comprising placing the bait preparation according to any one of claims 1 to 16 in the habitat, wherein the antigen elicits a protective response against the infection.

18. The method according to claim 17, wherein the bait preparation is deployed by air.

19. Use of the bait preparation according to any one of claims 1 to 16 for eliciting a protective immune response against a pathogen in wild or feral pigs.

20. The use according to claim 19, wherein the bait preparation is deployed by air.

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