Method and use for inhibiting African swine fever virus and / or classical swine fever virus in animal feed or animal feed ingredients

Abstract

The present application relates to methods and uses for inhibiting African swine fever virus and / or classical swine fever virus in animal feed or animal feed ingredients. The present invention provides a method of inhibiting African swine fever virus and / or classical swine fever virus in an animal feed or an animal feed ingredient, comprising: introducing a chemical slow-epidemic agent into the feed or feed ingredient, the chemical slow-epidemic agent comprising medium chain fatty acid and / or essential oil, wherein the chemical slow-epidemic agent is introduced at a content of from about 0.125% to less than 2% by weight based on the total weight of the animal feed or feed ingredient considered as 100% by weight. The chemical slow-epidemic agent is effective when introduced into a feed or feed ingredient at a much lower inclusion rate than existing methods for inhibiting other microorganisms. The method is particularly suitable for the post-processing of animal feed, feed ingredients or pet food for transportation and storage for several days or weeks.

Description

[0001] This application is a divisional application of the Chinese patent application with the application date of March 1, 2019, application number "2019800166262", and invention name "Chemical Mitigation of African Swine Fever Virus and Classical Swine Fever Virus".

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of priority to U.S. Provisional Patent Application Serial No. 62 / 637,825, filed on March 2, 2018, entitled “INACTIVATION OF VIRUSES SUCH AS AFRICAN SWINE FEVER VIRUS (ASFV) AND CLASSICAL SWINE FEVER VIRUS (CSFV) WITH MEDIUM CHAIN ​​FATTYACIDS,” and U.S. Provisional Patent Application Serial No. 62 / 780,740, filed on December 17, 2018, entitled “CHEMICAL MITIGATION OF AFRICAN SWINE FEVER VIRUS,” each of which is incorporated herein by reference in its entirety. Technical Field

[0004] The present invention broadly relates to methods of inhibiting African swine fever virus and / or classical swine fever virus in animal feed, feed ingredients, and pet foods. Background Art

[0005] Medium-chain fatty acids have been shown to be effective against specific domestic pathogens, porcine epidemic diarrhea virus (PEDV) and Salmonella sp. (U.S. Patent Application Publication No. 2017 / 0354167, published on December 14, 2017, which is incorporated herein by reference in its entirety).

[0006] African swine fever virus and classical swine fever virus are foreign animal diseases known to be transmitted by the oral route. Specifically, African swine fever virus (ASFV) is a very large complex DNA virus that is rapidly spreading in China, the world's largest pork producer. ASFV causes high mortality in pigs and is currently a foreign animal disease in North America and most European countries. There is currently no effective vaccine, and the virus is known to be transmitted by the oral route. ASFV can survive in feed and feed ingredients that experience various environmental conditions that simulate transoceanic transport. ASFV is a very unique double-stranded DNA virus and is the only virus in the African swine fever virus family (Asfarviridae) and the African swine fever virus genus (Asfivirus). Importantly, there is no suitable alternative virus to ASFV. Classical swine fever virus (CSFV) is a single-stranded RNA virus in the Flaviviridae family (Flaviviridae). Porcine epidemic diarrhea virus (PEDV) is not related to any of these viruses and belongs to the Coronaviridae family (Coronaviridae). The genome sizes of these three viruses vary significantly, with PEDV measuring 28 kb compared to CSFV's 12.3 kb and ASFV's 190 kb. Viruses are known to vary widely in their stability in the environment and their sensitivity to disinfectants. Even viruses within the same family can exhibit varying inactivation rates, and these three viruses do not belong to the same family. Published studies comparing the stability of several viruses that cause foreign animal diseases have demonstrated significant variation. Additionally, other previously published work has demonstrated significant variation in the stability of feed ingredients between PEDV, ASFV, and bovine viral diarrhea virus (BVDV), a virus in the same family and used as a surrogate for CSFV. Variability in feed ingredient stability would predict differences in sensitivity to MCFAs and mitigation. Therefore, without direct evidence for mitigants against the viruses themselves, the effectiveness of mitigants against other viruses or bacteria cannot be extended or translated to ASFV or CSFV.

[0007] What is needed are treatments that effectively mitigate (e.g., inactivate) ASFV and CSFV in animal feed and feed ingredients, while also being safe for oral administration to pigs and other animals. Summary of the Invention

[0008] This application describes a method for inhibiting African swine fever virus and / or classical swine fever virus in animal feed or animal feed ingredients. The method comprises introducing a chemical retarder into the feed or feed ingredient. The chemical retarder comprises (consists essentially of, or even consists of): a medium-chain fatty acid and / or an essential oil, and is introduced into the feed or feed ingredient at an inclusion rate of less than 2% by weight (but typically at least about 0.125% by weight), based on the total weight of the animal feed or feed ingredient, taken as 100% by weight.

[0009] In another embodiment, a chemical mitigating agent is provided for inhibiting African swine fever virus and / or classical swine fever virus in animal feed or animal feed ingredients. The chemical mitigating agent comprises medium-chain fatty acids and / or essential oils.

[0010] Also described herein are treated animal feeds and / or animal feed ingredients that are resistant to African swine fever virus and / or classical swine fever virus. Based on the total weight of the animal feed or feed ingredient considered as 100% by weight, the feed or feed ingredient includes about 0.125% to less than 2% by weight of a chemical retardant. Chemical retardants include medium-chain fatty acids and / or essential oils. Exemplary treated animal feeds or animal feed ingredients for the purposes of the present invention include complete pig feed, blood meal, pork bone meal (MBM), spray-dried animal plasma, feather meal, poultry blood meal, poultry by-product meal, vitamin D, lysine hydrochloride, choline chloride, soy meal, dry pet coarse food, and mixtures thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0012] Figure 1 is a graph showing dose response inactivation curves for ASFV (strain BA71V) exposed to varying concentrations of a 1:1:1 MCFA blend, wherein data are shown as titers following exposure to MCFA concentrations ranging from 0.125% to 2.0%, and as percent reduction in viral concentration compared to a positive control;

[0013] Figure 2 is a series of graphs showing detection of the ASFV Georgia 2007 genome over the course of a 30-day cross-border model, with data presented as mean cycle threshold (Ct) values ​​in duplicate at 1, 8, 17, and 30 days post-inoculation; and

[0014] Figure 3 is a graph showing the amount of ASFV DNA measured by qPCR at the end of the 30-day cross-border model in untreated controls (open bars) and in samples treated with MCFA at 28 dpi (black bars);

[0015] Figure 4 is a graph showing dose response inactivation curves for CSFV (Brescia isolate) exposed to varying concentrations of a 1:1:1 MCFA C6:C8:C10 blend, wherein data are shown as titers following exposure to MCFA concentrations ranging from 0.125% to 2.0%, and as percent reduction in viral concentration compared to a positive control; and

[0016] Figure 5A Positive control images from an experiment using indirect fluorescent antibody detection of CSFV Brescia on porcine kidney cells exposed or not to MCFA;

[0017] Figure 5B are images from experiments using indirect fluorescent antibody detection of CSFV Brescia on porcine kidney cells treated with 0.625% MCFA; and

[0018] Figure 5C Negative control images from experiments using indirect fluorescent antibody detection of CSFV Brescia on porcine kidney cells exposed or not to MCFA. DETAILED DESCRIPTION

[0019] The present invention generally relates to methods for inhibiting African swine fever virus (ASFV) and / or classical swine fever virus (CSFV) in animal feed, feed ingredients, and pet food. More specifically, the present invention relates to chemical retardants for use in inhibiting ASFV and / or CSFV in various types of animal and pet food ingredients, as well as complete feed diets and pet food products. In general, chemical retardants include medium-chain fatty acids and / or essential oils. As used herein, "inhibit" or "inhibition" refers to a reduction in the measurable level of a target microorganism (i.e., ASFV or CSFV) or a reduction in the growth rate of the microorganism as compared to an untreated control. In one or more embodiments, an effective amount of a chemical retardant is used according to the methods of the present invention to inhibit ASFV and / or CSFV in animal feed or animal feed ingredients, for example, to a concentration below the level of detection by RT-PCR and / or virus isolation in cell culture. As used herein, "effective amount" refers to an amount of an active compound (e.g., medium-chain fatty acids and / or essential oil) that provides a bioavailable level sufficient to achieve a desired property improvement. In a preferred embodiment, the method according to the invention is advantageously applicable to the transport and storage of animal feed ingredients.

[0020] The chemical retardant used in one or more embodiments of the present invention may include medium-chain fatty acids and / or essential oils. In one or more embodiments, the chemical retardant includes (consists essentially of or even consists of): a medium-chain fatty acid, and more specifically, at least one medium-chain fatty acid. Medium-chain fatty acids are acids with aliphatic tails of 6 to 12 carbon atoms. In one or more embodiments, the medium-chain fatty acids used in the present invention include caproic acid, caprylic acid, capric acid, and / or lauric acid. Therefore, in certain embodiments, the chemical retardant is selected from the group consisting of caproic acid, caprylic acid, capric acid, lauric acid, and mixtures thereof. However, in certain other embodiments, the chemical retardant does not contain lauric acid. Therefore, in such embodiments, the chemical retardant may be selected from the group consisting of caproic acid, caprylic acid, capric acid, and mixtures thereof. In one or more embodiments, a blend of medium-chain fatty acids may be used. For example, in one or more embodiments, a blend of two or more medium-chain fatty acids may be introduced into the feed or feed ingredients. In one or more embodiments, a blend of caproic acid, caprylic acid, and capric acid is introduced into a feed or feed ingredient in a weight ratio of about 1:1:1 (equal parts). This combination increases the solubility of the medium-chain fatty acids and effectively inactivates viruses and improves pig growth when administered orally. However, blends containing other weight ratios of caproic acid, caprylic acid, and capric acid may be used within the scope of the present invention.

[0021] In one or more embodiments, the chemical mitigating agent includes (consists essentially of or even consists of): essential oils, and more specifically at least one essential oil. Essential oils are concentrated hydrophobic liquids containing volatile aromatic compounds derived from plants. There are a variety of different essential oils that can be used in one or more embodiments of the present invention. A non-exclusive list of these essential oils includes: agar oil, ajwain oil, angelica root oil, fennel oil, asafoetida, Peru balsam, basil oil, laurel oil, bergamot oil, black pepper, buchu oil, birch, camphor, wormwood oil, cardamom seed oil, carrot seed oil, cedar oil, chamomile oil, calamus root, cinnamon oil, labdanum species, citron, citronella oil, sage, clove oil, coffee, coriander, tansy oil (tansy leaf oil) , Costus Root, Cranberry Seed Oil, Cubeb, Cumin Oil / Black Seed Oil, Cypress, Sedge, Curry Leaf, Caraway Oil, Dill Oil, Elecampane, Eucalyptus Oil, Cumin Seed Oil, Fenugreek Oil, Fir Wood, Frankincense Oil, Galangal, Maple, Geranium Oil, Ginger Oil, Eucalyptus Globulus, Grapefruit Oil, Henna Oil, Helichrysum, Hickory Nut Oil, Horseradish Oil, Hyssop, Idaho Tansy, Jasmine Oil, Juniper Berry Oil, Laurus nobilis), lavender oil, Ledum, lemon oil, lemongrass, lime, Litsea cubeba oil, agarwood (Linaloe), citrus, marjoram, Melaleuca See Tea tree oil, honey leaf oil, wild mint (Mentha arvensis) oil / peppermint oil, moringa oil, winter savory (Mountain Savory), artemisia oil, mustard oil (essential oil), myrrh oil, myrtle, neem oil or neem oil, neroli oil, nutmeg, orange oil, oregano oil, orris oil, sandalwood, celery oil, patchouli oil, perilla essential oil, mint oil, peppermint oil, petitgrain, pine oil, ravensar leaf, red juniper, Roman chamomile, rose oil, rosehip oil, rosemary oil, rosewood oil, sage oil, sandalwood oil, sassafras oil, savory oil oil), schisandra oil, spearmint oil, spikenard, spruce, star anise oil, citrus, tarragon oil, tea tree oil, thyme oil, hemlock, turmeric, valerian, vetiver oil (vetiver oil), western red cedar, wintergreen, yarrow oil, ylang ylang and zedoary. In one or more embodiments, the chemical mitigating agent is selected from the group consisting of garlic oleoresin, turmeric oleoresin, capsicum oleoresin, rosemary extract, wild oregano essential oil and mixtures thereof. In one or more embodiments, a blend of essential oils can be used. For example, in one or more embodiments, a blend of two or more essential oils can be introduced into the feed or feed ingredients. In one or more embodiments, a blend of essential oils comprising equal parts of garlic oleoresin, turmeric oleoresin, capsicum oleoresin, rosemary extract and wild oregano essential oil is introduced into the feed or feed ingredients.However, blends comprising other weight ratios of garlic oleoresin, turmeric oleoresin, capsicum oleoresin, rosemary extract, and wild oregano essential oil may be used within the scope of the present invention.

[0022] In one or more embodiments, a mixture comprising (consisting essentially of or even consisting of) medium-chain fatty acids and essential oils, and more specifically a blend of one or more medium-chain fatty acids and one or more essential oils, may be used as a chemical retardant. However, in certain other embodiments, no essential oils or other types of retardants are used in addition to the medium-chain fatty acids. In addition, embodiments of the present invention avoid the use of toxic chemicals, such as formaldehyde, which are not introduced into animal feed, feed ingredients, or pet food. Therefore, in one or more embodiments, the chemical retardant consists essentially of (or even consists of) one or more medium-chain fatty acids. In certain preferred embodiments, the chemical retardant consists essentially of (or even consists of) a blend of caproic acid, caprylic acid, and capric acid.

[0023] While any effective amount of chemical retardant can be used, in one or more embodiments, the chemical retardant is introduced into the animal feed (including pet food) or feed ingredient at an inclusion rate of about 0.01% to about 10% by weight, preferably about 0.05% to about 5% by weight, more preferably about 0.1% to about 2% by weight, and most preferably about 0.5% to about 0.9% by weight, based on the total weight of the feed or feed ingredient considered as 100% by weight. Advantageously, the chemical retardants described herein, and in particular the medium-chain fatty acids, are effective inactivators of ASFV and CSFV (i.e., 4-log reduction) at inclusion rates as low as 0.6% by weight (for ASFV) and 0.5% by weight (for CSFV), which are amounts far lower than doses shown to be effective against other microorganisms in the prior art. Thus, in one or more embodiments, the chemical retarder is introduced into the animal feed or feed ingredient at an inclusion rate of less than 2% by weight, less than 1.5% by weight, less than 1% by weight, less than 0.9% by weight, less than 0.8% by weight, less than 0.7% by weight, or less than 0.6% by weight, based on the total weight of the feed or feed ingredient considered as 100% by weight. In one or more embodiments, lower doses may also be used, for example, when a 4-log reduction in ASFV or CSFV is not desired. In one or more such embodiments, the chemical retarder may be introduced into the animal feed (including pet food) or feed ingredient at an inclusion rate of about 0.125% by weight to about 0.5% by weight, based on the total weight of the feed or feed ingredient considered as 100% by weight. Thus, in certain other embodiments, the chemical retardant is introduced into the animal feed or feed ingredient at an inclusion rate of at least 0.125 wt%, at least 0.25 wt%, at least 0.5 wt%, at least 0.6 wt%, at least 0.7 wt%, at least 0.8 wt%, at least 0.9 wt% or at least 1 wt%, based on the total weight of the feed or feed ingredient considered as 100 wt%.

[0024] In one or more embodiments, in addition to medium-chain fatty acids and / or essential oils, sodium bisulfate can also be added to animal feed or feed ingredients. Sodium bisulfate is an acid salt that is considered "generally recognized as safe" (GRAS) and a "natural product" by the FDA. In one or more embodiments, sodium bisulfate can be dissolved in a solution and applied to the surface of an animal food or food ingredient to prevent or reduce bacterial growth. In one or more embodiments, a sodium bisulfate solution is applied to the surface of a dry pet food or pet food ingredient. For example, the solution can be applied to the surface of dry dog ​​food (kibble) or dry cat food. In one or more embodiments, based on the total weight of the feed or feed ingredient considered to be 100% by weight, the solution can be applied to the surface of the feed or ingredient so as to provide sodium bisulfate at an inclusion rate of about 0.1% by weight to about 2% by weight, more preferably about 0.15% by weight to about 1.5% by weight, and even more preferably about 0.2% by weight to about 1% by weight.

[0025] The chemical retardants used according to the present invention can be used to treat a variety of animal feeds or animal feed ingredients. However, in one or more embodiments, the methods according to the present invention are particularly suitable for use with pig feed and feed ingredients. In such embodiments, the animal feed or animal feed ingredient can be selected from the group consisting of: a complete pig diet, blood meal, pork bone meal (MBM), and spray-dried animal plasma. In one or more embodiments, the animal feed ingredient can include an ingredient selected from the group consisting of: vitamin D, lysine hydrochloride, choline chloride, and soy meal. In other embodiments, the chemical retardants can be used with pet food and pet food ingredients. In one or more embodiments, the pet food and pet food ingredients include dry dog ​​food (kibble) and / or cat food. The term "pet food" means any composition intended for consumption by a pet, and "dry" food, in the art, generally refers to pet food with a moisture content of less than about 20% (preferably less than about 15%, more preferably less than about 10%). The term "kibble" is used in the art to refer to particles of dry pet food.

[0026] The method according to one or more embodiments of the present invention can be used to produce animal or pet feed. Therefore, in one embodiment of the present invention, animal feed or pet food is provided, based on the total weight of the feed considered as 100% by weight, which contains about 0.01% by weight to about 10% by weight, more preferably about 0.05% by weight to about 5% by weight, even more preferably about 0.1% by weight to about 2% by weight, and most preferably about 0.5% by weight to about 0.9% by weight of a chemical retarder or a blend of chemical retardants (such as the chemical retardants and blends described herein). In one or more embodiments, animal feed or pet food is provided, based on the total weight of the feed considered as 100% by weight, which contains less than 2% by weight, less than 1.5% by weight, less than 1% by weight, less than 0.9% by weight, less than 0.8% by weight, less than 0.7% by weight or less than 0.6% by weight of a chemical retarder or a blend of chemical retardants (such as the chemical retardants and blends described herein). In one or more embodiments, an animal feed or pet food is provided, comprising from about 0.125% to about 0.5% by weight of a chemical retarder or a blend of chemical retardants (e.g., the chemical retardants and blends described herein), based on the total weight of the feed considered as 100% by weight. In one or more embodiments, an animal feed or pet food is provided, comprising at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, or at least 1% by weight of a chemical retarder or a blend of chemical retardants (e.g., the chemical retardants and blends described herein), based on the total weight of the feed considered as 100% by weight.

[0027] Embodiments of the present invention are particularly well suited for use in the transportation, particularly international transportation and storage, of feed and feed ingredients. Prior to the present invention, medium-chain fatty acids had not been shown to be effective mitigants for diseases, particularly viruses, that are foreign to U.S. industry. Feed or ingredients may become contaminated with ASFV and / or CSFV during processing. Advantageously, embodiments of the present invention are particularly well suited for inactivating and inhibiting the spread of these contaminants. Thus, in one or more embodiments, the method according to the present invention comprises introducing a chemical mitigant into the feed or feed ingredient after processing. The chemical mitigant may be mixed with the feed or feed ingredient for a sufficient time to provide a uniform mixture. In one or more embodiments, the method according to the present invention can prevent or reduce ASFV and / or CSFV in feed and / or ingredients during transportation and storage for at least about 90 days after processing, transportation and storage for at least about 60 days after processing, transportation and storage for at least about 40 days after processing, or transportation and storage for at least about 30 days after processing.

[0028] Embodiments of the present invention advantageously provide a safe alternative method for preventing or reducing ASFV and / or CSFV in animal or pet feed and ingredients. Existing methods using hazardous chemicals have been shown to negatively impact the animal's protein and amino acid metabolism. Unlike existing methods, the present invention utilizes substantially harmless chemical mitigants at doses found to achieve effective mitigation of ASFV and / or CSFV. The chemical mitigants used according to the present invention are natural alternatives that pose no substantial risk to worker or environmental safety.

[0029] After reading the disclosure herein and the following feasibility examples, other advantages of the various embodiments of the present invention will be apparent to those skilled in the art. It will be understood that, unless otherwise noted herein, the various embodiments described herein are not necessarily mutually exclusive. For example, the features described or depicted in one embodiment may also be included in other embodiments, but are not necessarily included. Therefore, the present invention encompasses various combinations and / or integration of the specific embodiments described herein.

[0030] As used herein, the phrase "and / or," when used in the context of a list of two or more items, means that any one of the listed items can be employed alone or any combination of two or more of the listed items can be employed. For example, if a composition is described as containing or excluding components A, B, and / or C, the composition can contain or exclude A alone; B alone; C alone; a combination of A and B; a combination of A and C; a combination of B and C; or a combination of A, B, and C.

[0031] This specification also uses numerical ranges to quantify certain parameters associated with various embodiments of the present invention. It should be understood that when numerical ranges are provided, these ranges will be interpreted as providing literal support for claim limitations that state only the lower values ​​of the range, as well as claim limitations that state only the higher values ​​of the range. For example, a disclosed numerical range of about 10 to about 100 provides literal support for a claim that lists "greater than or equal to about 10" (with no upper limit) and a claim that lists "less than or equal to about 100" (with no lower limit).

[0032] Example

[0033] The following examples illustrate the effectiveness of chemical mitigation strategies against ASFV and CSFV in feed and feed ingredients. However, it should be understood that these examples are provided for illustration only and nothing herein should be construed as limiting the overall scope of the present invention.

[0034] Protocols and procedures were developed for diluting and mixing various concentrations of medium-chain fatty acid blends (MCFA) with ASFV (BA71V isolate) and CSFV (Brescia isolate). This work has been performed in their respective cell cultures, green monkey kidney cells (vera cells) and porcine kidney cells. As a first step, MCFA were prepared by mixing equal volumes of C6:C8:C10 (hexanoic acid:octanoic acid:decanoic acid) in a volume ratio of 1:1:1. MCFA treatments were then prepared at concentrations ranging from 10% to 0.625% and mixed with a standard high concentration of virus (10 6 TCID 50 MCFA were mixed with 1% (0.625 mL / ml) of MCFA. MCFA were shown to be non-destructive to the cell cultures used in these experiments, and positive and negative controls were included in each test. Preliminary results indicate that MCFA were effective in inactivating both CSFV and ASFV at all tested doses, ranging from 10% to 0.625%.

[0035] Example 1

[0036] African swine fever virus testing

[0037] A specific protocol and procedure was used to dilute and mix various concentrations of medium-chain fatty acid blends in 20% DMSO with ASFV (BA71v isolate) in vero cells. As a first step, medium-chain fatty acids were prepared by mixing equal volumes of C6:C8:C10 in 20% DMSO at a volume ratio of 1:1:1. Medium-chain fatty acids were then prepared at concentrations ranging from 2% to 0.125% and mixed with a standard high concentration of ASFV (10 6 TCID 50 A positive control was included in each assay to determine the dose-response inactivation of the virus.

[0038] Results (Table 1, Figure 1 ) showed that at all tested doses from 0.7% to 2.0%, medium-chain fatty acid treatment effectively inactivated ASFV to levels undetectable by indirect fluorescent antibody testing. A dose-dependent reduction in ASFV was observed at medium-chain fatty acid concentrations ranging from 0.6% to 0.125%. At the lowest concentration of medium-chain fatty acids tested in cell culture (0.125% medium-chain fatty acids), approximately 0.75 log 10 TCID 50 / ml reduction in viral titer. At 0.25% medium-chain fatty acid inclusion, viral titer was reduced by approximately 98.2%. At 0.6% medium-chain fatty acid inclusion, an approximately 4-log reduction in viral titer was seen. A 4-log reduction in viral titer is the standard for viral inactivation as described by the World Organization for Animal Health (OIE). In summary, we have demonstrated that medium-chain fatty acids are effective inactivators of ASFV in cell culture, and the dose required for an approximately 4-log reduction is 0.6%, which is far lower than the standard 1% inclusion.

[0039]

[0040] ASFV survival at 1% (wt%) MCFA inclusion in 9 high-risk ingredients was also tested in 30 cross-border models simulating varying environmental temperature and humidity conditions using the ASFV Georgia 2007 isolate. ASFV Georgia 2007 is a highly virulent ASFV isolate currently circulating in China. High-risk feed ingredients included conventional soy meal, organic soy meal, soy cake, choline, moist cat food, moist dog food, dry dog ​​food, pork sausage casings, and complete feed. Detection and quantification of ASFV DNA were performed by qPCR and compared between untreated inoculated feed and MCFA-treated inoculated feed. In the first study, feed was treated with 1% MCFA containing C6:C8:C10 in a 1:1:1 ratio immediately before ASFV inoculation at 0 days post-inoculation (dpi). PCR results showed that all untreated control samples and 0 dpi MCFA-treated samples were positive for ASFV DNA at 1, 8, 17, and 30 days ( Figure 2 ).exist Figure 2 Data are shown for untreated controls (open squares) and samples treated with MCFA at 0 dpi, immediately before ASFV inoculation (black squares). All samples had detectable ASFV DNA at the end of the 30-day cross-border model. Ct values ​​>40 were considered negative. Note: PCR detects viral DNA but not infectivity.

[0041] In the second study, the detection and quantification of ASFV DNA were compared between untreated inoculated feed and inoculated feed that had been treated with 1% MCFA in a 1:1:1 ratio at 28 dpi. The results showed that all untreated and 28 dpi MCFA-treated samples were positive for ASFV DNA at 30 dpi ( Figure 3 ).exist Figure 3 Data are shown as the mean cycle threshold (Ct) of duplicates. All samples were positive for ASFV DNA at the end of the cross-border model. Ct values ​​>40 were considered negative.

[0042] Untreated inoculated feed and inoculated feed treated with 1% medium-chain fatty acids at 0 and 28 dpi were then tested by virus isolation on porcine alveolar macrophages to determine whether ASFV DNA detected by PCR was infectious. Virus isolation determined that infectious virus was present in all untreated positive controls, while infectious virus was undetectable in any samples treated with medium-chain fatty acids at 0 or 28 dpi (Table 2). Infectious virus was detected in positive untreated samples using a monoclonal antibody against the ASFV p30 protein.

[0043]

[0044] Samples treated with medium-chain fatty acids at 0 dpi or 28 dpi were then further tested in a young pig bioassay model to assess the presence of infectious virus. Samples from medium-chain fatty acid-treated feed were injected intramuscularly because this is the most sensitive method for detecting infectious ASFV. Pigs were injected with 1 or 2 samples to reduce the number of pigs used. Samples were combined based on quantitative PCR results. In the pig bioassay, all feed samples treated with medium-chain fatty acids at 0 dpi were negative for infectious ASFV (Table 3). In the pig bioassay, all feed samples except two treated with medium-chain fatty acids at 28 dpi were negative for infectious ASFV (Table 3). The two feed samples (one or both of which may have contained infectious ASFV) were organic soy flour and dry dog ​​food. These two samples were injected into a single pig, and ASFV was detected in the spleen of the pig during virus isolation. Overall, our data support that medium-chain fatty acids are effective mitigants for infectious ASFV in cell culture and feed ingredients.

[0045]

[0046] Example II

[0047] Classical swine fever virus detection

[0048] The effects of MCFA on CSFV in cell culture are shown in Table 4, and Figure 4 and Figures 5A to 5C Different levels of medium-chain fatty acid treatment (C6:C8:C10 at a 1:1:1 ratio) were tested for their efficacy in inactivating or reducing viral titers of a CSFV Brescia isolate in a cell culture model. MCFA levels tested ranging from 10% to 0.5% reduced CSF viral titers to levels below detectable by an indirect fluorescent antibody test on porcine kidney cells. Figure 5BThe results show that there was no detectable CSFV after MCFA treatment. A dose-dependent reduction in CSFV viral titers was demonstrated after exposure to MCFA levels ranging from 0.4% to 0.125%. The lowest MCFA inclusion rate tested (0.125%) resulted in an 82.2% reduction in viral titers when compared to the untreated positive control.

[0049]

[0050] The effect of MCFA on CSFV survival in feed ingredients subjected to cross-border environmental conditions was also tested. The ability of a 1% MCFA blend (C6:C8:C10 in a 1:1:1 ratio) to inactivate CSFV in two feed ingredients that supported CSFV survival in a 37-day cross-border model simulating transport was tested. The two ingredients tested included conventional soybean meal and pork sausage casings. 5 TCID 50 Before 1 dpi, the MCFA blend was added to the feed ingredients and vortexed to mix the MCFA throughout the feed ingredients. For conventional soy flour and pork sausage casings, samples treated with MCFA were negative for virus isolation and titration as early as 1 dpi, while the positive controls had 10 4.3 TCID 50 and 10 3.7 TCID 50 Detectable CSFV titers were achieved. All samples (including the untreated positive control) were negative for virus isolation and titration up to 37 dpi. Supernatants from traditional soybean meal samples treated with and without MCFA were collected at the end of the 37-day cross-border model and tested in a 3-week-old pig bioassay by intramuscular injection. Untreated soybean meal samples were positive for CSFV in the pig bioassay, while soybean meal samples treated with MCFA were negative for CSFV in the pig bioassay, demonstrating the efficacy of MCFA in eliminating CSFV infectivity in soybean meal subjected to cross-border transport conditions.

Claims

1. A method for inhibiting African swine fever virus and / or classical swine fever virus in animal feed or animal feed ingredients, the method comprising: introducing a chemical retarder into the feed or feed ingredients, the chemical retarder comprising a medium chain fatty acid and / or an essential oil, The chemical retardant is introduced at a content rate of about 0.125 wt % to less than 2 wt % based on the total weight of the animal feed or feed ingredient regarded as 100 wt %.

2. The method of claim 1, wherein the medium chain fatty acid is selected from the group consisting of caproic acid, caprylic acid, capric acid, lauric acid, and mixtures thereof.

3. The method according to claim 1 or 2, wherein the chemical mitigant is a blend of two or more medium-chain fatty acids.

4. The method of any one of claims 1 to 3, wherein the chemical mitigant comprises a blend of medium-chain fatty acids comprising caproic acid, caprylic acid, and capric acid.

5. The method of any one of claims 1 to 4, wherein the chemical mitigant comprises a blend of medium-chain fatty acids comprising approximately equal parts caproic acid, caprylic acid, and capric acid.

6. The method according to any one of claims 1 to 5, wherein the essential oil is selected from the group consisting of garlic oleoresin, turmeric oleoresin, capsicum oleoresin, rosemary extract, wild oregano essential oil, and mixtures thereof.

7. The method of any one of claims 1 to 6, wherein the chemical mitigant comprises a blend of medium chain fatty acids and essential oils.

8. The method of any one of claims 1 to 7, wherein the animal feed or animal feed ingredient is selected from the group consisting of: complete swine feed, blood meal, pork bone meal (MBM), spray-dried animal plasma, feather meal, poultry blood meal, poultry by-product meal, vitamin D, lysine hydrochloride, choline chloride, and soy meal.

9. The method of any one of claims 1 to 7, wherein the animal feed or animal feed ingredient is dry pet kibble, the method further comprising applying sodium bisulfate to the surface of the dry pet kibble.

10. The method of claim 9, wherein the sodium bisulfate is applied to the surface of the dry pet kibble at an inclusion rate of about 0.1 wt% to about 2 wt%, based on the total weight of the animal feed or feed ingredient considered as 100 wt%.

11. Use of chemical mitigants comprising medium chain fatty acids and / or essential oils for inhibiting African swine fever virus and / or classical swine fever virus in animal feed or animal feed ingredients.

12. The use according to claim 11, wherein the medium chain fatty acid is selected from the group consisting of caproic acid, caprylic acid, capric acid, lauric acid and mixtures thereof.

13. The use according to claim 11 or 12, wherein the chemical mitigating agent is a blend of two or more medium-chain fatty acids.

14. The use according to any one of claims 11 to 13, wherein the chemical mitigating agent comprises a blend of medium-chain fatty acids comprising caproic acid, caprylic acid and capric acid.

15. The use according to any one of claims 11 to 14, wherein the chemical mitigant comprises a blend of medium chain fatty acids comprising approximately equal parts caproic acid, caprylic acid and capric acid.

16. Use according to any one of claims 11 to 15, wherein the essential oil is selected from the group consisting of garlic oleoresin, turmeric oleoresin, capsicum oleoresin, rosemary extract, wild oregano essential oil and mixtures thereof.

17. The use according to any one of claims 11 to 16, wherein the chemical mitigant comprises a blend of medium chain fatty acids and essential oils.

18. The use according to any one of claims 11 to 17, wherein the chemical mitigating agent comprises a blend of essential oils comprising approximately equal parts garlic oleoresin, turmeric oleoresin, capsicum oleoresin, rosemary extract and wild oregano essential oil.

19. Use of a chemical retarder comprising medium-chain fatty acids and / or essential oils in the preparation of a treated animal feed or animal feed ingredient for protection against African swine fever virus and / or classical swine fever virus, wherein the treated animal feed or animal feed ingredient comprises from about 0.125 wt% to less than 2 wt% of the chemical retarder, based on the total weight of the animal feed or feed ingredient considered as 100 wt%, wherein the animal feed or animal feed ingredient is selected from the group consisting of: complete swine feed, blood meal, pork bone meal (MBM), spray-dried animal plasma, feather meal, poultry blood meal, poultry by-product meal, vitamin D, lysine hydrochloride, choline chloride, soy meal, dry pet kibble, and mixtures thereof.

20. The use of claim 19, wherein the animal feed or animal feed ingredient is dry pet kibble, further comprising sodium bisulfate on the surface of the dry pet kibble.

Images