Bait vaccines

Abstract

The instant disclosure provides a bait vaccine formulation for swine, the formulation comprising an antigen; optionally, an adjuvant, and a matrix, wherein said antigen is inside of a container, wherein said container is entirely within the matrix, wherein the matrix is not coated by a protective film, wherein said bait formulation is temperature stable and / or humidity stable and / or suitable for aerial deployment. Methods of using these bait vaccine formulations are also provided.

Description

BAIT VACCINESTHE NAMES OF THE PARTIES TO A JOINT RESEARCH AGREEMENT

[0001] The claimed invention was made as a result of activities undertaken within the scope of a joint research agreement between Zoetis LLC and IREC (UCLM-CSIC).FIELD OF THE INVENTION

[0002] This invention is generally in the field of bait vaccines for the protection of wild boars.BACKGROUND

[0003] Disease control in wildlife represents a notable challenge (Delahay et aL, 2009) and oral vaccination is one of a handful tools available to combat the most serious infectious diseases shared with wildlife (Gortazar et al., 2015). It is used to reduce the number of animals that are susceptible below the threshold for the maintenance of the infection (Rupprecht et al., 2004; Blancou et al., 2009), or to diminish disease severity and, thereby, infection spread (Garrido et al., 2011).

[0004] The Eurasian wild boar (Sus scrofa) is a natural reservoir of several pathogens shared with humans and with farm animals, and therefore effective disease control is necessary to mitigate the consequences caused by 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 (Muller et al., 2012). In Europe, and in the specific case of wild boar, first field tests were carried out in the 1990s to control Classical Swine Fever (CSF) and oral vaccination of wild boar against CSF was later expanded to different European countries (Delegated regulation UE 2020 / 689). For this process, different factors must be considered, such as an early deployment of vaccination, the structure of the landscape and spatial distribution of food sources, and professional support to work alongside hunters and wildlife and forestry agencies (Rossi et al., 2015).

[0005] Vaccines can seek contact with the oropharyngeal mucosa (Ballesteros et al., 2007) or seek to cross the gastric barrier without the acids and enzymes inactivating the antigen, to reach the intestine. The latter can be achieved in two ways, one by lipid composition baits (Aldwell etal., 2003), as fats are attacked by bile acids in the small intestine, and alternatively by capsules or other protective containers that dissolve in the intestine (Mahato et al., 2003). African swine fever vaccines would most likely target the oral mucosa (Sang et al., 2020).

[0006] Effective oral vaccination of wildlife requires the development of baits that are effective for oral administration of vaccines, stable, and preferably host specific (Brauer et aL, 2006, Ballesteros et al., 2007). This task is particularly difficult for piglets from 2 to 4 months of age, the ideal age for CSF vaccination (Brauer et al., 2006). Precedent baits include the RIEMSER® one for classical swine fever vaccination (Kaden et al., 2000), the Australian PIGOUT® Feral Pig Bait used for delivery of toxicants to feral pigs in Australia and the USA (Cowled et al., 2006), and the IREC bait used for oral vaccination against animal tuberculosis in Spain (Ballesteros et al., 2009).

[0007] However, these baits have certain drawbacks. RIEMSER® baits have a low melting point (30°C), and its shape and texture can make the bait difficult to handle and lead to vaccine loss (Rossi et al., 2015). In addition, this bait is too large to be consumed by wild boars under4 months of age (Faust et al., 2007). The PIGOUT® bait requires high temperatures in the extrusion process for its production (Beltran-Beck et al. 2013).

[0008] 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 51-53°C) as cement and were found suitable for field application during summer with relatively high temperatures (up to 44?C, Beltran-Beck et al., 2014). However, these baits have a limited resistance to humidity (Ballesteros et al., 2009) and are not species-specific (Ballesteros et al., 2011; Beltran-Beck et al., 2014).

[0009] Furthermore, the low uptake of baits by piglets younger than 6 months has been a constant problem in previous wild boar vaccination attempts due to the low vaccination rates at that age (Brauer et al., 2006, Rossi et al., 2011, Sage et al., 2011, Calenge and Rossi, 2014). Field trials with the IREC bait achieved up to 92%% bait uptake by piglets, mostly by using piglet- targeted selective feeders (Ballesteros et aL, 2011, Diez-Delgado et al., 2019).

[0010] An efficient and specific bait deployment strategy for the species to be vaccinated is one of the most daunting challenges since there are multiple non-target species such as birds or carnivores that can interfere with bait uptake. To prevent birds from consuming baits, a problemthat also occurs in the use of rodenticides for pest control, the possibility of using some sort of colorant as a deterrent has been studied, verifying the effectiveness of the use of blue and green colors to reduce bait consumption by birds (Cowan et al., 2017).

[0011] Baits have been administered through numerous deployment strategies such as by air from planes or helicopters (Kaden et al., 2002), burying them to specifically target wild boar and protect the live vaccine from damage due to high temperatures (Kaden et al., 2002), through selective piglet feeders (Ballesteros et aL, 2009), or deploying the baits under heavy stones which wild boar can lift (Diez-Delgado et al., 2019). Airborne bait deployment is particularly suited for interventions over large areas (Siers et al., 2017). However, I REC type baits have never been deployed from aircraft and assessments of bait resistance are lacking.

[0012] Accordingly, there is a need in the art for improved baits for suid (wild boar and pig) vaccination.SUMMARY OF INVENTION

[0013] This disclosure addresses these and other needs in the art by providing, in the first aspect, a bait vaccine formulation for swine, the formulation comprising: a. an antigen, b. optionally, an adjuvant, c. a matrix, wherein said antigen is inside of a container, wherein said container is entirely within the matrix, wherein the matrix is not coated by a protective film, wherein said bait formulation is i) temperature stable and / or ii) humidity stable and / or iii) suitable for aerial deployment.

[0014] In certain embodiments, the matrix comprises corn flour, piglet feed, sugar, and a binder, wherein said piglet 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 soy flour, 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 gluten, and from about 1.5% to about 3% w / w fatty acids. Preferably, the piglet feed comprises about 37% w / w barley, about 32% w / w wheat, about 10.5% w / w soy flour,about 6% W / \N peas, about 5% w / w wheat bran, about 4% w / w corn gluten, about 2.3% w / w fatty acids, and further comprises about 1.2% w / w di-calcium 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, said corn flour is present at about 14% w / w to about 22% w / w of said matrix, said piglet feed is present at about 35% w / w to about 45% w / w of said matrix, said sugar is present at about 12% w / w to about 18 % w / w of said matrix, and said binder is present at about 24% w / w to about 28 % w / w of said matrix. In other preferred embodiments, wherein said corn flour is present at about 17% w / w of said matrix, said piglet feed is present at 40-42% w / w of said matrix, said sugar is present at 14-16% w / w of said matrix, and said binder is present at 24- 26% w / w of said matrix.

[0015] In certain embodiments, applicable to any of the compositions recited above, the binder is honey or sugar cane. In further additional or alternative embodiments, also applicable to any of the compositions recited above the sugar is sucrose.

[0016] Preferably, in the bait of any of the embodiments recited above, the antigen is inside a container, and wherein said container is entirely within the matrix.

[0017] In certain embodiments, the bait is generally oblong shape, wherein the longest dimension is at least 1.8 times greater than the second-longest dimension and wherein said longest dimension is between about 2.5 cm and about 6 cm. In more preferred embodiments, said bait vaccine formulation has a generally semi-spherical 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.

[0018] In certain embodiments of the invention applicable to any bait formulation recited above, said formulation is weight-stable at 25-42 °C.

[0019] In further additional or alternative embodiments the invention applicable to any bait formulation recited above, said formulation is humidity-stable.

[0020] In further additional or alternative embodiments the invention applicable to any bait formulation recited above, said formulation is suitable for aerial deployment.

[0021] In certain embodiments applicable to any of the formulations recited herein, the antigen is selected from the group consisting of antigens protecting from infections selected from the group consisting of African swine fever virus, classical wine fever virus, Aujeszky's disease virusand Mycobacterium tuberculosis complex and any combination thereof. In more preferred embodiments, the antigen is an ASF antigen.

[0022] In certain embodiments applicable to any formulations recited herein, said bait vaccine formulation is black, green, or blue-colored.

[0023] In the second aspect, the instant disclosure provides a method of eliciting protective immune response against a pathogen in a wild boar or feral pig population in a habitat, the method comprising placing the bait formulation of according to any of the embodiments of the first aspect of the invention in said habitat, wherein the antigen elicits protective response from said infection. In certain embodiments of this second aspect of the invention, said bait formulations are deployed aerially.

[0024] In the third aspect, the instant disclosure provides a use of the bait formulation according to any embodiments of the first aspect for eliciting protective immune response against a pathogen in a wild boar or feral pig. In certain embodiments of this third aspect, said bait formulation is deployed aerially.DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0025] The terms "about" or "approximately", when used in connection with a measurable numerical variable, refer to the indicated value of the variable and to all values of the variable that are within the experimental error of the indicated value (e.g., within the 95% confidence interval for the mean) or within 10 percent of the indicated value, whichever is greater.

[0026] The term "antigen" refers to any substance that is recognized by the animal's immune system and generates an immune response. The term includes killed, inactivated, attenuated, or modified live bacteria, viruses, or parasites. The term "antigen" also includes polynucleotides, polypeptides, recombinant proteins, synthetic peptides, protein extract, cells (including tumor cells), tissues, polysaccharides, or lipids, or fragments thereof, individually or in any combination thereof. The term antigen also includes antibodies, such as anti-idiotype antibodies or fragments thereof, and to synthetic peptide mimotopes that can mimic an antigen or antigenic determinant (epitope).

[0027] The term "humidity-stable" refers to changes in weight of the bait formulation after said bait formulation was no more than 7% submerged into water for 72 hours at 25°C to 42°C. Thus, the formulation is humidity stable if under the conditions above its weight changed by no more than 5% compared to the weight before the test. In preferred embodiments, the weight of the humidity-stable formulation changes by no more than 4%, or no more than 3%, or no more than 2%, or no more than 1%.

[0028] The term "oblong shape" refers to shapes having a longer and shorter dimension and where the cross section is about the same or less than the shorter dimension. Preferably, the longest dimension is at least 1.8 times greater than the second-longest dimension. In the more preferred embodiments, the longer dimension is from 1.8 to 2.5 times greater than the second longer dimension, including, without limitations 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 an easier consumption. Without limitations, the oblong shape may be in a form of a prism, cylinder, or ellipsoid shape. In certain preferred embodiments, the oblong shape is hemispherical, with dimensions of about 2.5 to about 4 cm by about 1 to about 2 cm.

[0029] The phrase "suitable for aerial deployment" refers to displacement of less than 8 mm, measured as provided in the Example.

[0030] The term "pathogen" is used together with the antigen. Briefly, the antigen is capable of eliciting the protective immune response against the respective pathogen.

[0031] The term "protective immune response" refers to a reduction in an intensity or a duration of at least one clinical symptom of an infection with a given pathogen. Clinical symptoms vary depending on the nature of the pathogen and may include, without limitations, fever, diarrhea, lung lesions, nasal shedding, oral shedding fecal shedding, and other symptoms. Preferably, the protective immune response prevents the infection of the vaccinated animal with the pathogen. The protective immune response may be measured directly or inferred by immunological endpoints, including markers of cell-mediated immune system as well as protective antibody titers.

[0032] The term "vaccine" refers to a composition containing an antigen, wherein the composition elicits the protective immune response against the respective pathogen.

[0033] In a broad aspect, the invention provides a bait vaccine formulation for swine, the formulation comprising: an antigen; optionally, an adjuvant; and a matrix, wherein said antigen is inside of a container, wherein said container is entirely within the matrix, wherein the matrix is not coated by a protective film, wherein said bait formulation is temperature-stable and / or humidity-stable and / or suitable for aerial deployment.

[0034] The matrix of the bait comprises corn flour, piglet feed, sugar, and a binder. Preferably, the binder is honey or sugar cane molasses. More preferably, the binder is honey. Generally, said corn flour is present at about 14% w / w to about 22% w / w of said matrix, said piglet feed is present at about 35% w / w to about 45% w / w of said matrix, said sugar is present at about 12% w / w to about 18 % w / w of said matrix, and said binder is present at about 24% w / w to about 28 % w / w of said matrix.

[0035] In other embodiments, said corn flour is present at about 14% w / w to 21% w / w of said matrix, said piglet feed is present at about 35% w / w to 43% w / w of said matrix, said sugar is present at about 12% w / w to about 18 % w / w of said matrix, and said binder (e.g., honey) is present at about 24% w / w to about 28 % w / w of said matrix.

[0036] In further preferred embodiments, wherein said corn flour is present at about 17% w / w of said matrix, said piglet feed is present at 40-42% w / w of said matrix, said sugar is present at 14-16% w / w of said matrix, and honey is present at 24-26% w / w of said matrix.

[0037] In any of the matrix compositions described above, the piglet 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 soy flour, 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 gluten, and from about 1.5% to about 3% w / w fatty acids. It should be understood that the discussion of “w / w" percentage in application to the piglet feed, the percentages are given in relationship 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.

[0038] In more specific embodiments, also suitable for any matrix composition described above, the piglet feed comprises about 37% w / w barley, about 32% w / w wheat, about 10.5% w / w soy flour, about 6% w / w peas, about 5% w / w wheat bran, about 4% w / w corn gluten, about 2.3% w / w fatty acids, and further comprises about 1.2% w / w di-calcium phosphate, about 1.1% w / w calcium carbonate, about 0.5% w / w minerals, vitamin A, vitamin D3, copper, lysine, and sodium chloride (about 0.4% w / w).

[0039] Natural beeswax has been used as a superhydrophobic coating (Li et aL, 2018). However, the inventors observed that wild boar tended to break coated baits, consuming the exposed matrix. This is undesired because it might lead to the loss of the Eppendorf tube and therefore a vaccination failure. Accordingly, it is preferred that the baits disclosed herein do not contain any coatings.

[0040] In a particularly preferred embodiment, in said matrix the corn flour is present at about 17% w / w of said matrix, said sugar is present at 14-16% w / w of said matrix, honey is present at 24.5-25.5% w / w of said matrix, and said piglet feed is present at 40-42% w / w of said matrix, wherein said piglet feed comprises about 37% w / w barley, about 32% w / w wheat, about 10.5% w / w soy flour, about 6% w / w peas, about 5% w / w wheat bran, about 4% w / w corn gluten, about 2.3% w / w fatty acids, and further comprises about 1.2% w / w di-calcium phosphate, about 1.1% w / w calcium carbonate, about 0.5% w / w minerals, vitamin A, vitamin D3, copper, lysine, and about 0.4% sodium chloride.

[0041] The baits of the invention are generally oblong, including cylindrical, semicylindrical, hemispheric, ellipsoid, oval, hemi-ellipsoid, prism-shape and so on. It is preferred that the angles of these shapes are rounded for an easier consumption. It is preferred that the bait should be generally hemispheric. The dimensions of the bait are limited by the size of the animals. It is currently preferred that the long dimension should be about 2.5 to about 6 cm, and the shorter dimension should be 1.8-2.5 times less than the longer dimension. In the most preferred embodiments, the bait is of a hemispheric shape, 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 embodiments, the bait is of a hemispheric shape, 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.

[0042] The baits of the invention generally possess at least one of the following properties -they are temperature-resistant, they are humidity-resistant, and / or they are suitable for aerial deployment. In more preferred embodiments, at least two of these properties are present, and in even more preferred embodiments, all three properties are present. The temperature-stable baits of the invention do not change weight after 72 hours at a temperature ranging from 25°C to 42°C, and particularly at the temperature ranging from 37°C to 42 °C. The humidity-stable baits of the invention do not change weight after being about 7% submerged in water for up to 72 hours. In particularly preferred embodiments, the exposure to temperature of 4°C to 42°C for up to 48°C and humidity (submerging, as described in the example) do not affect the impact resistance of the baits of the invention.

[0043] The matrix of the bait contains the container which holds the antigen(s) and optional adjuvant(s). It is preferred that the container be fully within the matrix. The volume of the container should be sufficient to contain the antigen(s) and the optional adjuvant(s). In practice, the volume of the container may vary between about 1 ml and about 0.1 ml, including, without limitations, 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 baits described herein, the volume of the container is about 0.2 ml.

[0044] Containers may be biodegradable or not biodegradable. Suitable non-limiting materials for the container include without limitations cellulose, PLA / PGA polymers, and plastic. In certain embodiments, the container is EPPENDORF® vial.

[0045] Multiple antigens are suitable for administration via baits. The most suitable antigens can elicit the protective immune response against pathogens that affect wild boar and feral pig populations and that can be transmitted to farmed pigs. More specifically, the pathogens include, without limitations, African Swine Fever virus, Classical Swine Fever virus, Pseudorabies virus. In other embodiments, the pathogen may be of bacterial or protozoan origin, including, without limitations, members of the Mycobacterium tuberculosis complex and other mycobacteria, Lawsonia intracellularis, giardia, Cryptosporidium spp, and other pathogens.

[0046] Both monovalent vaccines (antigens against one pathogen) and multivalent vaccines (antigens against multiple pathogens) may be present in the container. The antigens may bepresent in the form of inactivated pathogens (for example, inactivated bacteria, inactivated viruses, bacterins), modified live pathogens, subunits and DNA vaccines. The combinations of these antigens are also possible.

[0047] The container may also include optional adjuvants. Multiple adjuvants are known in the field and the choice of the adjuvant would depend on the nature of the antigens. Suitable adjuvants include, without limitations, Aluminum compounds, mycobacterium cell walls, saponin (including its complexes with sterols and / or phospholipids such as, for example ISCOM®), lipopolysaccharides, poly l:C, CpG-containing immunostimulatory oligonucleotides, glycolipids, liposomes. Different combinations of these adjuvants are also possible.

[0048] In addition to the antigens and the optional adjuvants, various modifications are possible for the bait vaccine formulations suitable for administration with the baits recited herein. For example, the contents of the container may include mucoadhesives, carriers, antibiotics, antiparasitic drugs, other medicaments, buffers, preservatives and the like.

[0049] The baits of the invention may be deployed by multiple methods. For example, in the habitats where the wild pigs' diets are supplemented by feed, such as corn or piglet feed, the baits may be left with the corn or the piglet feed. In other embodiments, the baits may be left in places known to be frequented by pigs, e.g., near farm fields or on pigs' tracks. Other methods, such as areal deployment, deployment in piglet-selective feeding cages, and deployment under heavy stones, are also possible.

[0050] Aerial bait deployment has proven successful in fox rabies control in Europe (Muller et al., 2012). Baiting from aircraft has also been used to control certain species with toxic baits, 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, none of the previously existing wild boar baits has been tested for their suitability for aerial deployment. As described in the examples, the inventors simulated areal deployment using compression tests thus measuring the displacement that could be caused by a collusion of the bait with the ground. When the displacements were greater than 8 mm, it was considered that the impact plane collided with the container comprising the antigen and the optional adjuvant. This collision could lead to fracture. Considering this limit, it wasobserved that the new bait formulation resists impact from a maximum altitude of 500 meters. Thus, in different embodiments, the bait formulation is resistant to being dispersed from 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 or about 200 to about 300 meters above the ground.

[0051] The following example is presented as an illustrative embodiment but should not be taken as limiting the scope of the invention. Many changes, variations, modifications, and other uses and applications of this invention will be apparent to those skilled in the art.EXAMPLESEXAMPLE 1: Composition and Properties of the Bait

[0052] Materials and Methods

[0053] Use of animals in bait palatability trials

[0054] This research was performed using non-invasive photo-trapping and no animals were captured, handled, or sampled. The protocol was designed by specifically trained and certified scientists according to the EC Directive 86 / 609 / EEC and approved by the Animal Experiment Committee of Casti I la-La Mancha University and the Regional Ethic Committee (PR-2022-01-01).

[0055] Bait production and composition

[0056] The starting point was the IREC bait, which was designed to contain 0.2 ml polyethylene capsules to introduce the vaccine formulation. Baits were prepared with a matrix containing 44% piglet feed, 22% wheat flour, 16.5% paraffin (Dilabo SA, Madrid, Spain), 16.5% sucrose, and 1% cinnamon and truffle attractant in 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 endurance to ambient temperature and humidity. Ultimately, the improved bait matrix composition included 41% piglet feed (Piensos Inalsa, Ciudad Real, Spain), 17% corn flour, 15% sucrose, and 25% honey. Honey acted as cement instead of using paraffin. Both the IREC and the new bait contain, in the center of the bait matrix, a 200 pl Eppendorf tube (VWR, Pennsylvania, USA) to carry the target vaccine or substance.

[0057] Palatability trials

[0058] Palatability trials were performed in the field to rule out some particularities in the making of these baits. The experiments examined the use of protective coating (wax-coated baits vs. plain baits), different shapes (spheric vs. semispheric), composition (IREC formulation proposed by Ballesteros et al. (2009) vs. other formulae), a variety of aromas: vanilla-cinnamon-truffle (Norel S.A., Madrid, Spain), anise (PME Cake, Riverwalk Business Park, UK), almond (PME Cake, Riverwalk Business Park, UK), cadaverine (>97.0% GC, Sigma-Aldrich, Darmstadt, Germany); and colorants (black, green and blue; Wilton Brands LLC, Illinois, USA) to come up with the most suitable composition (Table 1). These tests were carried out by direct observation or using camera traps (Browning model BTC-5HDPX, Birmingham, USA) in a hunting estate with wild boar with total or partial freedom, whose diet was additionally supplemented with corn. Detection time (Td, time in which wild boar detect each bait group), and consumption time (Tc, time in which the wild boar fully consumes each bait group) were calculated.

[0059] Only camera traps were used for the field tests (10 tests including field experiments where birds gain access to baits). In these last field tests (bird field tests) 4 groups of different colors of five baits each are compared. The baits were distributed one in front of another, so that the five hidden cameras (placed for 48 hours) would capture the bait-type preference of wild boar and non-target species, respectively. During each test, cameras recorded the number of visits by each species and the proportion of baits consumed. Visits were filmed for 48 hours straight, capturing everything until baits would completely run out. A 10-minute interval was considered as defining a new visit.

[0060] Bait physical stability

[0061] Humidity

[0062] To quantify the effect of humidity on the bait's physical stability, three different groups of baits, consisting of 14 baits in total, were separately exposed to 3 conditions: a saturated medium (with the highest percentage of relative humidity that it can have, 100% at 25-C) (Hl), a saturated medium, but with 1 mm of water depth (medium in which the baits stay semisubmerged, 100% at 252C) (H2), and another medium where the baits are totally submerged (15^C) (H3). Samples were taken at 0, 10, 24, 34, 48, 58, and 72-hours. For comparison, 3 groupsof IREC baits (8 baits per group) were tested at the same conditions. Samples were taken at 0, 24, 48, and 72 hours.

[0063] At the indicated sampling time, the baits were weighed and allowed to fall from an altitude of 5 meters hitting on a concrete surface. The procedure was repeated up to 15 attempts per bait, and the number of impacts against the ground that the baits withstood without losing the Eppendorf tubes was recorded.

[0064] Temperature

[0065] Four different groups of 14 baits each were produced and exposed to 4° C, 25° C, 37° C, and 42^ c, respectively. Samples were taken at 0, 10, 24, 34, 48, 58, and 72-hours. For comparison, 4 groups of 8 IREC baits each were exposed to the same temperatures as above and samples were collected at 0, 24, 48, and 72-hours.

[0066] Simulations of aerial bait deployment

[0067] Simulations were run using SolidWorks CAD software (SolidWorks Corp., Dassault Systemes, Suresnes, France). Compression tests were carried out using an electro-mechanical universal testing machine from the Instron company (Instron 5696, Illinois Tool Works Inc., Glenview, Illinois, United States), a device equipped with a 1 kN load cell and compressive platens. The load is applied with a constant displacement rate of 5 mm / min. The specimens used for this test are cylindrical in shape, made of the same material as the baits, with a height L = 60 mm and a diameter D = 44 mm. During testing, a 3D Digital Image Correlation (DIC) system commercialized by Correlated Solutions Inc. (Irmo, SC, United States) was used for optical measurement of material displacements and strains. The DIC parameters used for these tests were: subset size = 85 pixels; step size = 11 pixels. Once the displacement field was obtained through the correlation of images of specimen during testing acquired each 4 seconds, the strains were derived thanks to the subsequent processing of the data using the VIC 3D software (Correlated Solutions Inc., Irmo, SC, United States). The stress was obtained as:where F is the applied load and A - n(D / 2)2is the initial cross section. Once the strains were obtained using the DIC technique, the Poisson ratio (v) was obtained as the ratio between thestrains £yyand sxx, which are the strains in the transverse (y-direction) and longitudinal (x- direction) averaged in the region of interest:

[0068] The elastic modulus (Young's modulus) is the slope of the stress-strain curve in the linear region, calculated by linear regression. Finally, the yielding strength was obtained as the stress level at the onset of nonlinearity in the stress-strain curve.

[0069] Statistical analysis

[0070] To perform the statistical analysis of the palatability tests, the Mann-Whitney U test was selected alongside the Kruskal-Wallis H test for the scrutiny of the wax used, in order to account for more than two groups, as recorded in R (R Core Team, 2018). In doing so, the results obtained during the physical stability tests for humidity and temperature showed that the variable "weight gain" and the variable "number of impacts" were both considered dependent variables. Similarly, the effect of composition and treatment during the 72 hours-range were noted. A GLM with a complete factorial model was proposed using IBM SPSS Statistics 24 (Chicago, Illinois, United States).

[0071] Results

[0072] Palatability tests

[0073] Table 1 presents the results of seven palatability trials with captive wild boar. While none of the trials resulted in statistically significant differences between prototypes, several relevant insights were gained. First, importantly, the average times to bait detection and bait consumption were almost identical regardless of the bait matrix tested, IREC vs. new formulation, indicating that the new formulation would not reduce bait uptake rates. Baits were (expectably) detected at the same time regardless their shape, but it took the wild boar double the time to consume the spherical baits, which tended to roll away. Furthermore, wax coating presence / absence and bait flavor and color were irrelevant for bait detection and consumption by wild boar. The cadaverine flavor was not tested because it was difficult to introduce in the bait matrix.

[0074] Table 1 summarizes palatability trials in captive wild boar. Results of seven trials are presented, testing the effects of the presence / absence of a wax coating, spherical vs.hemispherical bait shape, different flavors (vs. plain baits), and black coloring (vs. no colorant), indicating the number of baits per trial group, number of repeats, and average time to detection and time to consumption, in minutes, with their standard error.Table 1. Effect of Bait Color, Aroma, Shape, Matrix Composition on Bait Consumption

[0075] Field trials of bait preference

[0076] Camera traps were placed for 48 hours and the visits of different animal species, as well as the percentage of bait consumption per species were recorded (Table 2). A total of 96 visits to the baits was recorded, of which 49% were by corvids and 19% by the target species, wild boar. Regarding bait consumption, wild boar consumed 94% of the baits they approached, while foxes, dogs and birds consumed 38-42%.Table 2.- Species visiting baits (in %) and bait consumption rates per species (in %) during field trials with the new bait formulation. Tria s consisted of 10 baiting sites with 20 baits per site.

[0077] To deter birds, different dyes were tested to record the effect of bait color on bait preference by corvids. Results are synthetized in Table 3. In four of the trials a preference for non-colored baits was observed.Table 3.- Birds and their bait preferences as assessed in five field trials comparing baits with different color.

[0078] Except for birds, no animal shows preferences for the different colors used.

[0079] Physical stability

[0080] Physical stability trials regarding humidity and temperature compared the performance of the new bait formulation with the IREC bait.

[0081] Humidity

[0082] Baits completely submerged in water (extreme humidity, H3) dissolved after max 10 hours losing their shape regardless the bait type. Results of weight gain and resistance to dropping from 5 m altitude for baits placed in the saturated humidity environment (Hl) and in the saturated and semi-submerged environment (H2) and are shown in Table 4. For Hl, both bait types were found to absorb little moisture, remaining practically stable, whereas for H2 the IREC bait was heavily affected by moisture (Table 4; p>0.05). Regarding impact resistance, the new bait formulation outperformed the IREC bait. The IREC bait first increases its resistance when it is slightly moistened, but in a semi-submerged environment, where it absorbs more humidity, it soon ceases to be impact resistant (Table 4; p=0.036).Table 4: Comparison of the effects of humidity on bait weight and impact resistance between the new bait formulation and the IREC bait.

[0083] Temperature

[0084] As in the previous test, both bait types were subjected to different temperatures. The weights and the number of impacts that the baits resisted were recorded (Table 5). New formulation baits remained practically stable concerning temperature, while IREC baits were prone to weight loss, with a significant interaction between bait type and time (p=0.001). Regarding the impact resistance, new formulation baits were more resistant than IREC baits (Table 5; p>0.05).Table 5: Comparison of the effects of temperature on bait weight and impact resistance between the new bait formulation and the IREC bait.

[0085] Aerial bait delivery simulations

[0086] Through compressive tests and applying DIC technology, 8 specimens were tested, from which 5 representative testing outputs were obtained (Table 6).Table 6: Results of elastic modulus (Young's modulus) and yielding strength for the tests carried out.

[0087] Notice that the global longitudinal strain of the specimen £ = AL / L is calculated as the displacement of the actuatorZIL divided by the initial length of the cylinder / .. The representation of the stress-strain response using the global strain obtained from the displacement of the actuator allows qualitatively visualizing the full response of the material during testing. Meanwhile, the DIC strain measurements are used, in which the stress-strain evolution is represented until the visual macro-damage of the specimen permitted to extract trustable strain fields. The Poisson's ratio (v) in the linear region was close to 0.5 (0.499). With this information, the impact simulations were analysed with SolidWorks. Finally, the average number of the maximum deformations produced in the 5 representative samples as well as at each simulated altitude were recorded.Table 7.* Average and standard deviation of the maximum deformations produced in the 5 representative samples for each altitude.

[0088] Taken together, these data suggest that the bait disclosed herein is more temperature- and humidity stable than IREC bait, is suitable for aerial deployment and allows for a more species-specific consumption without sacrifices in palatability.

[0089] All publications cited in the specification, both patent publications and non-patent publications, are indicative of the level of skill of those skilled in the art to which this invention pertains. All these publications are herein fully incorporated by reference to the same extent as if each individual publication were specifically and individually indicated as being incorporated by reference.

[0090] Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present invention. It is therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present invention as defined by the following claims.

Claims

CLAIMS1. A bait vaccine formulation for swine, the formulation comprising: a. an antigen; b. optionally, an adjuvant, c. a matrix, wherein said antigen is inside of a container, wherein said container is entirely within the matrix, wherein the matrix is not coated by a protective film, wherein said bait formulation is i) temperature stable and / or ii) humidity stable and / or iii) suitable for aerial deployment.

2. The bait formulation of claim 1, wherein the matrix comprises corn flour, piglet feed, sugar, and a binder, wherein said piglet 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 soy flour, 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 gluten, and from about 1.5% to about 3% w / w fatty acids.

3. The bait formulation of claim 2, wherein the piglet feed comprises about 37% w / w barley, about 32% w / w wheat, about 10.5% w / w soy flour, about 6% w / w peas, about 5% w / w wheat bran, about 4% w / w corn gluten, about 2.3% w / w fatty acids, and further comprises about 1.2% w / w di-calcium phosphate, about 1.1% w / w calcium carbonate, about 0.5% w / w minerals, vitamin A, vitamin D3, copper, lysine, and sodium chloride.

4. The bait formulation of claim 2 or 3, wherein said corn flour is present at about 14% w / w to about 22% w / w of said matrix, said piglet feed is present at about 35% w / w to about 45% w / w of said matrix, said sugar is present at about 12% w / w to about 18 % w / w of said matrix, and said binder is present at about 24% w / w to about 28 % w / w of said matrix.

5. The bait formulation of any one of claims 2-4, wherein said corn flour is present at about 17% w / w of said matrix, said piglet feed is present at 40-42% w / w of said matrix, said sugar is present at 14-16% w / w of said matrix, and said binder is present at 24-26% w / w of said matrix.

6. The bait formulation of any one of claims 2-5, wherein the binder is honey or sugar cane.

7. The bait formulation of any one of claims 2-6 wherein said sugar is sucrose.

8. The bait formulation of any one of claims 1-7 wherein the antigen is inside a container, and wherein said container is entirely within the matrix.

9. The bait formulation of any one of claims 1- 8, wherein said bait formulation is generally oblong shape, wherein the longest dimension is at least 1.8 times greater than the second- longest dimension and wherein said longest dimension is between about 2.5 cm and about 6 cm.

10. The bait formulation of any one of claims 1-9 wherein said formulation is weight-stable at 25-42 °C.

11. The bait formulation of any one of claims 1-10, wherein said formulation is humiditystable.

12. The bait formulation of any one of claims 1-10 wherein said formulation is suitable for aerial deployment.

13. The bait formulation of any one of claims 1-12, wherein said bait vaccine formulation has a generally semi-spherical 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 formulation of any one of claims 1-13, wherein the antigen is an ASF antigen.

15. The bait formulation of any one of claims 1-14, wherein the antigen is selected from the group consisting of antigens protecting from infections selected from the group consisting of African swine fever virus, classical wine fever virus, Aujeszky's disease virus and Mycobacterium tuberculosis complex and any combination thereof.

16. The bait formulation of any one of claims 1-15, wherein said bait vaccine formulation is black, green, or blue-colored.

17. A method of eliciting protective immune response against a pathogen in a wild boar or feral pig population in a habitat, the method comprising placing the bait formulation of any one of claims 1-16 in said habitat, wherein the antigen elicits protective response from said infection.

18. The method of claim 17, wherein said bait formulations are deployed aerially.

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

20. The use according to claim 19, wherein said bait formulation is deployed aerially.

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