Bait Vaccine

Abstract

The present disclosure provides bait vaccine formulations for pigs, the formulation comprising an antigen, optionally an adjuvant, and a matrix, wherein the antigen is within a container, the container is entirely within the matrix, the matrix is ​​not coated with a protective film, and the bait formulation is temperature-stable and / or humidity-stable and / or suitable for airborne deployment. Methods of using these bait vaccine formulations are also provided.

Description

[Technical Field]

[0001] Names of the parties to the joint research agreement The invention as claimed has been made as a result of activities carried out within the scope of a collaborative research agreement between Zoetis LLC and IREC (UCLM-CSIC).

[0002] The present invention is generally in the field of bait vaccines for the protection of wild boars. [Background technology]

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

[0004] Eurasian wild boars (Sus scrofa) are natural reservoirs of several pathogens shared with humans and livestock, and therefore require effective disease control to mitigate the consequences of shared infections (Ballesteros et al., 2007). Oral vaccination has great potential to control infections in wild reservoirs and prevent outbreaks in other species (Mueller et al., 2012). In Europe, in the specific case of wild boars, the first field trials were conducted in the 1990s to control classical swine fever (CSF), and oral vaccination of wild boars against CSF was later expanded to different European countries (Delegated Regulation UE 2020 / 689). This process requires consideration of different factors, such as early vaccination deployment, the structure of the landscape and spatial distribution of food sources, and specialized support for collaboration with hunters and wildlife and forestry agencies (Rossi et al., 2015).

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

[0006] Effective oral vaccination of wildlife requires the development of baits that are effective, stable, and preferably host-specific for oral administration of vaccines (Brauer et al., 2006; Ballesteros et al., 2007). This task is particularly challenging for piglets aged 2 to 4 months, the ideal age for CSF vaccination (Brauer et al., 2006). Previous baits include RIEMSER®, a bait for classical swine fever vaccination (Kaden et al., 2000), the Australian PIGOUT® feral pig bait used for delivery of toxic substances to feral pigs in Australia and the United States (Cowled et al., 2006), and the IREC bait used for oral vaccination of animals against tuberculosis in Spain (Ballesteros et al., 2009).

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

[0008] The IREC baits proposed by Ballesteros et al. (2009) were designed to contain a 0.2 ml polyethylene capsule for the introduction of the vaccine formulation. These baits used paraffin (melting point 51–53°C) as a cement and were found to be suitable for field application during the summer months at relatively high temperatures (up to 44°C; Beltran-Beck et al., 2014). However, these baits have limited tolerance to humidity (Ballesteros et al., 2009) and are not species-specific (Ballesteros et al., 2011; Beltran-Beck et al., 2014).

[0009] Furthermore, low bait intake by piglets under 6 months of age has been a constant problem in previous wild boar vaccination attempts due to 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 IREC baits have achieved up to 92% bait intake by piglets by using selective feeders that primarily target piglets (Ballesteros et al., 2011, Diez-Delgado et al., 2019).

[0010] Developing an efficient and specific bait deployment strategy for the species being vaccinated is one of the most challenging tasks, as there are multiple non-target species, such as birds or carnivores, that may prevent bait consumption. To prevent birds from consuming the bait, a problem that also occurs when using rodenticides for pest control, the possibility of using some colorants as deterrents has been investigated, and the effectiveness of using blue and green colors to reduce bait consumption by birds has been examined (Cowan et al., 2017).

[0011] Baits have been administered through numerous deployment strategies, including by air from airplanes or helicopters (Kaden et al., 2002), by burying them to specifically target wild boars and protect the live vaccine from heat damage (Kaden et al., 2002), via selective piglet feeders (Ballesteros et al., 2009), or by deploying baits under heavy stones that wild boars can lift (Diez-Delgado et al., 2019). Aerial bait deployment is particularly suitable for widespread interventions (Siers et al., 2017). However, IREC-type baits have never been deployed from aircraft, and evaluation of bait resistance is lacking.

[0012] Therefore, there is a need in the art for improved baits for suid (wild boar and pig) vaccination. Summary of the Invention

[0013] The present disclosure provides in a first aspect a bait vaccine formulation for pigs, the formulation comprising: a. Antigen, b. optionally, an adjuvant; c. matrix, including the antigen is in a container, the container is completely within a matrix, the matrix is ​​not coated with a protective film, and the bait formulation i) be temperature stable, and / or ii) is humidity stable, and / or iii) Addressing these and other needs in the art by providing a bait vaccine formulation for swine that is suitable for aerial deployment.

[0014] In certain embodiments, the matrix comprises corn flour, piglet feed, sugar, and a binder, wherein 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% pea flour, 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 soybean flour, about 6% w / w pea flour, 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 dicalcium phosphate, about 1.1% w / w calcium carbonate, and about 0.5% w / w of the minerals vitamin A, vitamin D3, copper, lysine, and sodium chloride. More preferably, the corn flour is present at about 14% to about 22% w / w of the matrix, the piglet feed is present at about 35% to about 45% w / w of the matrix, the sugar is present at about 12% to about 18% w / w of the matrix, and the binder is present at about 24% to about 28% w / w of the matrix. In another preferred embodiment, the corn flour is present at about 17% w / w of the matrix, the piglet feed is present at 40-42% w / w of the matrix, the sugar is present at 14-16% w / w of the matrix, and the binder is present at 24-26% w / w of the matrix.

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

[0016] Preferably, in the bait of any of the above embodiments, the antigen is within a container, the container being entirely within the matrix.

[0017] In certain embodiments, the bait is generally rectangular, with the longest dimension being at least 1.8 times greater than the next longest dimension, and the longest dimension being from about 2.5 cm to about 6 cm. In more preferred embodiments, the bait vaccine formulation has a generally hemispherical shape, with the longer dimension being from about 2.5 cm to about 3.6 cm and the shorter dimension being from about 1 cm to about 1.7 cm.

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

[0019] In a further additional or alternative embodiment of the present invention applicable to any of the bait formulations described above, the formulation is humidity stable.

[0020] In a further additional or alternative embodiment of the present invention applicable to any of the bait formulations described above, the 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 that protect against infection 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. In a more preferred embodiment, the antigen is an ASF antigen.

[0022] In certain embodiments applicable to any of the formulations listed herein, the bait vaccine formulation is black, green, or blue.

[0023] In a second aspect, the present disclosure provides a method of inducing a protective immune response against a pathogen in a wild boar or feral pig population within a habitat, the method comprising placing within the habitat a bait formulation according to any of the embodiments of the first aspect of the invention, wherein the antigen induces a protective response from the infection. In certain embodiments of this second aspect of the invention, the bait formulation is deployed aerialy.

[0024] In a third aspect, the present disclosure provides a use of a bait formulation according to any embodiment of the first aspect to induce a protective immune response against a pathogen in wild boars or feral pigs. In certain embodiments of this third aspect, the bait formulation is deployed aerialy. DETAILED DESCRIPTION OF THE INVENTION

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

[0026] The term "antigen" refers to any substance that can be recognized by an animal's immune system and generate 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 extracts, 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-idiotypic antibodies or fragments thereof, and synthetic peptide mimotopes that can mimic antigens or antigenic determinants (epitopes).

[0027] The term "humidity stable" refers to the change in weight of a bait formulation after the bait formulation is immersed in water at 25°C to 42°C for 72 hours by no more than 7%. Thus, under these conditions, a formulation is humidity stable if its weight changes by no more than 5% compared to its weight before testing. In preferred embodiments, the weight of a 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 "rectangle" refers to a shape having a longer dimension and a shorter dimension, with a cross section approximately equal to or smaller than the shorter dimension. Preferably, the longest dimension is at least 1.8 times larger than the second longest dimension. In more preferred embodiments, the longer dimension is 1.8 to 2.5 times larger than the second longest dimension, including, but not limited to, about 1.9, 2, 2.1, 2.2, 2.3, and 2.4 times larger. Preferably, the ends of the rectangle are rounded for ease of consumption. Without limitation, the rectangle may be in the form of a prism, a cylinder, or an ellipse. In certain preferred embodiments, the rectangle is hemispherical, with dimensions of about 2.5 to about 4 cm by about 1 to about 2 cm.

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

[0030] The term "pathogen" is used in conjunction with antigens. Briefly, antigens are capable of eliciting a protective immune response against the respective pathogen.

[0031] The term "protective immune response" refers to a reduction in the intensity or duration of at least one clinical symptom of infection by a given pathogen. Clinical symptoms vary depending on the nature of the pathogen and may include, but are not limited to, fever, diarrhea, pulmonary lesions, nasal shedding, oral shedding, fecal shedding, and other symptoms. Preferably, a protective immune response prevents infection of the vaccinated animal with the pathogen. Protective immune responses may be measured directly or inferred by immunological endpoints, including markers of the cell-mediated immune system and protective antibody titers.

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

[0033] In a broad aspect, the present invention provides a bait vaccine formulation for pigs, the formulation comprising an antigen, optionally an adjuvant, and a matrix, wherein the antigen is within a container, the container is entirely within the matrix, and the matrix is ​​not coated with a protective film, and the bait formulation is temperature stable and / or humidity stable and / or suitable for airborne deployment.

[0034] The bait matrix 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 an amount of about 14% to about 22% w / w of the matrix, the piglet feed is present in an amount of about 35% to about 45% w / w of the matrix, the sugar is present in an amount of about 12% to about 18% w / w of the matrix, and the binder is present in an amount of about 24% to about 28% w / w of the matrix.

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

[0036] In a more preferred embodiment, the corn flour is present at about 17% w / w of the matrix, the piglet feed is present at 40-42% w / w of the matrix, the sugar is present at 14-16% w / w of the matrix, and the honey is present at 24-26% w / w of the matrix.

[0037] In any of the above matrix compositions, the piglet feed comprises about 30% w / w to about 42% w / w barley, about 30% w / w to about 35% w / w wheat, about 9% w / w to about 11% w / w soy flour, about 3% w / w to about 10% pea flour, about 2% w / w to about 8% wheat bran, about 1% w / w to about 7% w / w corn gluten, and about 1.5% to about 3% w / w fatty acids. It should be understood that the "w / w" percentages discussed in piglet feed applications are given with respect to the total composition of the piglet feed, not the total composition of the bait matrix. The piglet feed may also include minerals such as copper, vitamins (e.g., vitamin A and vitamin D), amino acids (such as polylysine), and sodium chloride.

[0038] In a more specific embodiment, which is also suitable for any of the matrix compositions 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 dicalcium phosphate, about 1.1% w / w calcium carbonate, about 0.5% w / w of the 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 boars tend to break the coated bait and consume the exposed matrix. This is undesirable because it can lead to the loss of the Eppendorf tube and therefore vaccination failure. Therefore, it is preferable that the bait disclosed herein does not contain any coating.

[0040] In a particularly preferred embodiment, the matrix comprises corn flour at about 17% w / w of the matrix, the sugar at 14-16% w / w of the matrix, honey at 24.5-25.5% w / w of the matrix, and the piglet feed at 40-42% w / w of the matrix, the piglet feed comprising about 37% w / w barley, about 32% w / w wheat, about 10.5% w / w soy flour, about 6% w / w pea, about 5% w / w wheat bran, about 4% w / w corn gluten, about 2.3% w / w fatty acids, and further comprising 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 about 0.4% sodium chloride.

[0041] The bait of the present invention is generally rectangular, including cylindrical, semi-cylindrical, hemispherical, elliptical, oval, semi-elliptical, prismatic, etc. The angles of these shapes are preferably rounded to facilitate consumption. The bait is preferably generally hemispherical. The dimensions of the bait are limited by the size of the animal. Currently, it is preferred that the long dimension be about 2.5 to about 6 cm, and that the short dimension be 1.8 to 2.5 times smaller than the long dimension. In the most preferred embodiment, the bait is hemispherical, with the longer dimension being about 2.9 to about 3.8 cm and the shorter dimension being about 1 to about 1.8 cm. In the most preferred embodiment, the bait is hemispherical, with the longer dimension being about 2.9 to about 3.5 cm and the shorter dimension being about 1 to about 1.5 cm.

[0042] Baits of the present invention generally have at least one of the following properties: they are heat-resistant, they are moisture-resistant, and / or they are suitable for airborne deployment. In more preferred embodiments, at least two of these properties are present, and in even more preferred embodiments, all three properties are present. Temperature-stable baits of the present invention do not change weight after 72 hours at temperatures ranging from 25°C to 42°C, particularly at temperatures ranging from 37°C to 42°C. Humidity-stable baits of the present invention do not change weight after immersion in water at about 7% humidity for up to 72 hours. In particularly preferred embodiments, exposure to temperatures of up to 48°C, 4°C to 42°C, and humidity (immersion, as described in the Examples) does not affect the impact resistance of the baits of the present invention.

[0043] The bait matrix includes a reservoir holding the antigen(s) and optional adjuvant(s). Preferably, the reservoir is completely within the matrix. The volume of the reservoir should be sufficient to contain the antigen(s) and optional adjuvant(s). In practice, the volume of the reservoir may vary from about 1 ml to 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, and about 0.9 ml. In one particular embodiment applicable to any bait described herein, the volume of the reservoir is about 0.2 ml.

[0044] The container may or may not be 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 an EPPENDORF® vial.

[0045] Multiple antigens are suitable for administration via bait. The most suitable antigens are capable of eliciting a protective immune response against pathogens that affect wild boar and feral pig populations and can be transmitted to farmed pigs. More specifically, pathogens include, but are not limited to, African swine fever virus, classical swine fever virus, and pseudorabies virus. In other embodiments, the pathogen may be of bacterial or protozoan origin, including, but not limited to, 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 polyvalent vaccines (antigens against multiple pathogens) can be present in the container. Antigens can be present in the form of inactivated pathogens (e.g., inactivated bacteria, inactivated viruses, bacteria), modified live pathogens, subunit, and DNA vaccines. Combinations of these antigens are also possible.

[0047] The container may also contain an optional adjuvant. Several 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 wall, saponin (including its complexes with sterols and / or phospholipids, such as ISCOM®), lipopolysaccharide, poly I:C, CpG-containing immunostimulatory oligonucleotides, glycolipids, and liposomes. Different combinations of these adjuvants are also possible.

[0048] In addition to the antigen and optional adjuvant, various modifications are possible to the bait vaccine formulation suitable for administration with the baits listed herein. For example, the contents of the container may include mucoadhesives, carriers, antibiotics, antiparasitic drugs, other agents, buffers, preservatives, etc.

[0049] The bait of the present invention can be deployed in several ways. For example, in habitats where the diet of feral pigs is supplemented with feed, such as corn or piglet feed, the bait can be left with the corn or piglet feed. In other embodiments, the bait can be left in locations known to be frequented by pigs, such as near farms or in pig tracks. Other methods are also possible, such as area deployment, deployment in piglet selective feeding cages, and deployment under heavy stones.

[0050] Aerial bait deployment has proven successful in controlling fox rabies in Europe (Mueller et al., 2012). Airborne baiting has also been used to control certain species using toxic baits, such as the possum (Trichosurus vulpecula) in New Zealand (Morgan, 2010), the feral cat (Felis silvestris) and red fox in Australia (Moseby et al., 2011), or the invasive brown tree snake (Boiga irregularis) in Guam (Goetz et al., 2021). However, none of the previously existing wild boar baits have been tested for their suitability for aerial deployment. As described in the Examples, we used a compression test to simulate area deployment and thus measure the displacement that can be caused by the bait's interaction with the ground. If the displacement exceeded 8 mm, the impact surface was considered to have collided with the container containing the antigen and optional adjuvant. This impact can lead to cracking. With this limitation in mind, the new bait formulation has been observed to withstand impacts from altitudes of up to 500 meters. Thus, in different embodiments, the bait formulation is resistant to being dispersed at about 100-500 meters above ground level, or about 200-500 meters above ground level, or about 300-500 meters above ground level, or about 400-500 meters above ground level, or about 100-400 meters above ground level, or about 200-300 meters above ground level.

[0051] The following examples are presented as illustrative embodiments and should not be construed as limiting the scope of the present invention. Many variations, modifications, and other uses and applications of the present invention will be apparent to those skilled in the art. [Example]

[0052] Example 1: Bait composition and characteristics Materials and Methods

[0053] Use of animals in bait palatability testing

[0054] This study was conducted using non-invasive phototrapping; animals were not captured, handled, or sampled. The protocol was designed by specially trained and certified scientists in accordance with EC Directive 86 / 609 / EEC and was approved by the Animal Experiments Committee and the Regional Ethics Committee of the University of Castilla-La Mancha (PR-2022-01-01).

[0053]

[0055] Bait production and composition

[0056] The starting point was the IREC bait, designed to contain a 0.2 ml polyethylene capsule for the introduction of the vaccine formulation. The bait was prepared with a matrix containing 44% piglet feed, 22% wheat flour, 16.5% paraffin (Dilabo SA, Madrid, Spain), 16.5% sucrose, and 1% cinnamon, as well as powdered truffle attractant (Norel SA, Madrid, Spain) (Ballesteros et al., 2009). Different components and protective films were tested to improve the bait's mechanical properties and its durability against ambient 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. The honey acted as a cement instead of using paraffin. Both the IREC and the new bait contain a 200 μl Eppendorf tube (VWR, Pennsylvania, USA) in the center of the bait matrix to carry the target vaccine or substance.

[0054]

[0057] Preference test

[0058] To rule out some of the peculiarities in the construction of these baits, palatability tests were conducted in the field. Experiments investigated the use of protective coatings (wax-coated baits vs. plain baits), different shapes (spherical vs. hemispherical), compositions (the IREC formulation proposed by Ballesteros et al. (2009) vs. other formulations), various scents: vanilla-cinnamon-truffle (Norel SA, 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 determine the most suitable composition (Table 1). These tests were conducted in fully or partially free wild boar hunting areas by direct observation or using camera traps (Browning model BTC-5HDPX, Birmingham, USA), whose diet was supplemented with corn. Detection time (Td, the time it took for wild boars to detect each bait group) and consumption time (Tc, the time it took for wild boars to completely consume each bait group) were calculated.

[0055]

[0059] For field trials (10 trials, including a field experiment in which birds accessed the bait), only camera traps were used. These final field trials (bird field trials) compared four groups of five differently colored baits each. The baits were distributed one before the other, so that five hidden cameras (placed for 48 h) captured the bait type preferences of wild boars and non-target species, respectively. During each trial, the cameras recorded the number of visits by each species and the proportion of bait consumed. Visits were filmed continuously for 48 h and recorded until the bait was completely depleted. A 10-min interval was considered to define a new visit.

[0056]

[0060] Physical stability of the bait

[0061] humidity

[0062] To quantify the effect of humidity on the physical stability of the bait, three different groups of baits, consisting of a total of 14 baits, were exposed to three conditions: saturated medium (the highest relative humidity percentage it can have, 100% at 25°C) (H1), saturated medium with 1 mm of water (medium in which the bait remained semi-submerged, 100% at 25°C) (H2), and another medium (15°C) in which the bait was fully submerged (H3). Samples were measured at 0, 10, 24, 34, 48, 58, and 72 hours. For comparison, three groups of IREC baits (eight baits per group) were tested under the same conditions. Samples were measured at 0, 24, 48, and 72 hours.

[0057]

[0063] At the indicated sampling times, the baits were weighed and dropped from a height of 5 meters onto a concrete surface. The procedure was repeated up to 15 attempts per bait, and the number of ground impacts the bait endured without losing the Eppendorf tube was recorded.

[0058]

[0064] temperature

[0065] Four different groups of 14 baits each were prepared and exposed to temperatures of 4°C, 25°C, 37°C, and 42°C, respectively. Samples were measured at 0, 10, 24, 34, 48, 58, and 72 hours. For comparison, four 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.

[0059]

[0066] Simulation of airborne bait deployment

[0067] Simulations were performed using SolidWorks CAD software (SolidWorks Corp., Dassault Systèmes, Suresnes, France). Compression tests were performed using an electromechanical universal testing machine (Instron 5696, Illinois Tool Works Inc., Glenview, Illinois, United States) equipped with a 1 kN load cell and 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 of L = 60 mm and a diameter of D = 44 mm. During the tests, a 3D Digital Image Correlation (DIC) system commercialized by Correlated Solutions Inc. (Irmo, SC, United States) was used for optical measurement of material displacement and strain. The DIC parameters used for these tests were subset size = 85 pixels and step size = 11 pixels. The displacement field was obtained through the correlation of images of the specimen during the test taken every 4 seconds, and the strain was derived thanks to subsequent processing of the data using VIC 3D software (Correlated Solutions Inc., Irmo, SC, United States). The stress was obtained as follows:

number

number

number

number

[0060]

[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 yield strength was obtained as the stress level at which the nonlinearity of the stress-strain curve begins.

[0061]

[0069] statistical analysis

[0070] To perform statistical analysis of the preference test, the Mann-Whitney U test was used in conjunction with the Kruskal-Wallis H test, as recorded in R (R Core Team, 2018). For the waxes used, three or more groups were selected for analysis. The results obtained during the physical stability test for humidity and temperature indicated that the variables "weight gain" and "number of impacts" were both considered dependent variables. Similarly, the effects of composition and treatment during the 72-hour period were noted. A GLM with a full factorial design was proposed using IBM SPSS Statistics 24 (Chicago, Illinois, United States).

[0062]

[0071] result

[0072] Preference test

[0073] The results of seven palatability tests with captive wild boars are shown in Table 1. While none of the tests yielded statistically significant differences between the prototypes, several relevant insights were obtained. First, and importantly, the mean times for bait detection and consumption were nearly identical regardless of the bait matrix tested, IREC versus the new formulation, indicating that the new formulation did not reduce bait intake rates. Although the baits were detected simultaneously regardless of shape (as expected), wild boars took twice as long to consume the spherical bait, which tended to rotate. Furthermore, the presence or absence of a wax coating, as well as the flavor and color of the bait, were unrelated to bait detection and consumption by wild boars. Cadaverine flavor was not tested because it was difficult to incorporate into the bait matrix.

[0063]

[0074] Table 1 summarizes the palatability tests in captive wild boars. Results from seven trials testing the effects of wax coating and non-wax coating, spherical vs. hemispherical bait shapes, different flavors (vs. plain bait), and black coloring (vs. no colorant) are presented, along with the number of baits per test group, the number of replicates, and the mean times to detection and consumption in minutes, along with their standard errors. [Table 1]

[0064]

[0075] Field test of bait preference

[0076] Camera traps were deployed for 48 hours to record visits by different animal species and the proportion of bait consumed by each species (Table 2). A total of 96 visits to the baits were recorded, of which 49% were by corvids and 19% by the target species, wild boars. Wild boars consumed 94% of the baits they approached, while foxes, dogs, and birds consumed 38–42%. [Table 2]

[0065]

[0077] To deter birds, different dyes were tested to record the effect of bait color on bait preference by corvids. The results are summarized in Table 3. Preference for the uncolored bait was observed in four of the tests. [Table 3]

[0066]

[0078] With the exception of birds, no animal shows a preference for different colors used.

[0067]

[0079] physical stability

[0080] Physical stability tests with respect to humidity and temperature compared the performance of the new bait formulation with IREC bait.

[0068]

[0081] humidity

[0082] Baits completely submerged in water (extreme humidity, H3) dissolved and lost their shape after up to 10 hours, regardless of bait type. Table 4 shows the weight gain and resistance to descent from a 5-m altitude for baits placed in saturated humidity environments (H1) and saturated and semi-submerged environments (H2). For H1, both bait types were found to absorb very little moisture and remain essentially stable, whereas for H2, the IREC bait was significantly affected by moisture (Table 4; p > 0.05). Regarding impact resistance, the new bait formulation outperformed the IREC bait. When slightly moistened, the IREC bait initially increased its resistance, but in the semi-submerged environment, where it absorbed more moisture, it quickly lost its impact resistance (Table 4; p = 0.036). [Table 4]

[0069]

[0083] temperature

[0084] As in the previous study, both bait types were subjected to different temperatures. The weight and number of impacts the bait resisted were recorded (Table 5). The new formulated bait remained essentially stable with respect to temperature, while the IREC bait was more susceptible to weight loss, with a significant interaction between bait type and time (p=0.001). With respect to impact resistance, the new formulated bait was more resistant than the IREC bait (Table 5; p>0.05). [Table 5-1] [Table 5-2]

[0070]

[0085] Aerial bait delivery simulation

[0086] Compression testing and DIC techniques were applied to test eight specimens from which five representative test outputs were obtained (Table 6). [Table 6]

[0071]

[0087] Overall longitudinal strain of the specimen

number

[0072]

[0088] Taken together, these data suggest that the baits disclosed herein are more temperature and humidity stable than IREC baits, are suitable for aerial deployment, and allow for more species-specific consumption without sacrificing palatability.

[0073]

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

[0074]

[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 can be made to the exemplary embodiments and that other arrangements can be devised without departing from the spirit and scope of the invention as defined by the following claims.

Claims

1. A bait vaccine formulation for pigs, comprising: a. antigen, b. optionally, an adjuvant; c. a matrix, the antigen is in a container, the container is completely within the matrix, the matrix is ​​not coated with a protective film, and the bait formulation i) is temperature stable, and / or ii) is humidity stable, and / or iii) A bait vaccine formulation for pigs that is suitable for aerial deployment.

2. 2. The bait formulation of claim 1, wherein the matrix comprises corn flour, piglet feed, sugar, and a binder, and 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% pea, 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. 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 soybean 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 dicalcium phosphate, about 1.1% w / w calcium carbonate, and about 0.5% w / w of the minerals vitamin A, vitamin D3, copper, lysine, and sodium chloride.

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

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

6. The bait formulation according to any one of claims 2 to 5, wherein the binder is honey or sugarcane.

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

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

9. 9. The bait formulation of any one of claims 1 to 8, wherein the bait formulation is generally rectangular with a longest dimension at least 1.8 times greater than the second longest dimension, the longest dimension being from about 2.5 cm to about 6 cm.

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

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

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

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

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

15. 15. The bait formulation of any one of claims 1 to 14, wherein the antigen is selected from the group consisting of antigens that protect against infection 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 to 15, wherein the bait vaccine formulation is black, green, or blue.

17. 17. A method of inducing a protective immune response against a pathogen in a wild boar or feral pig population within a habitat, said method comprising placing within said habitat a bait formulation according to any one of claims 1 to 16, wherein said antigen induces a response that protects against said infection.

18. 18. The method of claim 17, wherein the bait formulation is deployed aerially.

19. Use of a bait formulation according to any one of claims 1 to 16 for inducing a protective immune response against pathogens in wild boars or feral pigs.

20. 20. The use of claim 19, wherein the bait formulation is deployed in the air.

Images