Preventing virus transmission through feed

Abstract

The present invention relates to a computer-implemented method for determining the need to add a mitigating agent to feed to inactivate viruses that may be present in the feed. A preferred mitigating agent is a mixture comprising benzoic acid tablets and a powder comprising eugenol and thymol. The mixture rapidly inactivates viruses even when added only in low concentrations. This is particularly applicable to (but not limited to) porcine epidemic diarrhea virus (PEDV). Thus, by the present invention, the animal feed is substantially free of harmful viral RNA.

Description

Technical Field

[0001] The present invention addresses the threat of viral pathogens in feed. Background Art

[0002] Feed and feed ingredients have been proposed as contributing factors to the introduction of porcine epidemic diarrhea virus (PEDV) into commercial swine herds. This route of infection has been shown to be possible in experimental settings (Dee et al., “An evaluation of contaminated complete feed as a vehicle for porcine epidemic diarrhea virus infection of pigs following consumption via natural feeding behavior:proof of concept” BMC Veterinary Research, 2014; 10:176).

[0003] The Swine Health Information Center (SHIC), the National Pork Board, the National Pork Producers Council, and the American Association of Swine Veterinarians recommend adding more holding time to ensure viral degradation in feed. For conventional soybean meal, they recommend 143 days at 4°C to achieve 99.99% degradation (Swine Health Information Center, “Research on viral transmission in feedstuffs yields, new information” published on May 7, 2019).

[0004] WO 2011 / 017367 discloses an antimicrobial composition containing buffered propionic acid or acetic acid mixed with nonanoic acid. Embodiments of WO2016 / 081716 disclose animal feeds comprising a combination of about 0.01 wt % to about 5 wt % of a medium-chain fatty acid and an essential oil mixture. Gebhardt et al. evaluated spray-dried porcine plasma and porcine gestation diets containing 0.5% benzoic acid, 0.02% essential oil products, and combinations thereof (Gebhardt et al., "Determining the impact of commercial feed additives as potential porcineepidemic diarrhea virus mitigation strategies as determined by polymerasechain reaction analysis and bioassay", Translational Animal Science, January 1, 2019, (1): 134-142; citation in the introduction).

[0005] is a commercially available product that is claimed to combat viral pathogens in feed and / or ingredients. The supplier of this product recommends an inclusion rate of 4 kg per metric ton of feed, corresponding to 0.4% by weight. If a farmer follows this recommendation, feed costs increase by approximately $19.8 per metric ton of feed. Because the meat industry is very cost-sensitive, the additional cost of $19.8 per metric ton may be prohibitive.

[0006] A cheaper and / or more efficient solution is needed. Summary of the invention

[0007] When combating viral pathogens in feed, one would assume that a significant increase in the concentration of the mixture and / or an increase in the duration of treatment would increase the effectiveness of the treatment. This is not necessarily the case if a mixture of benzoic acid, eugenol and thymol is used.

[0008] To mitigate porcine epidemic diarrhea virus (PEDV), Gebhardt et al. teach the use of an essential oil product comprising 0.5% benzoic acid and 0.02%. Surprisingly, lower concentrations are sufficient if the essential oil used in combination with benzoic acid comprises eugenol and thymol. One embodiment of the present invention relates to a method for inhibiting porcine epidemic diarrhea virus in animal feed, wherein benzoic acid, eugenol and thymol are added to the animal feed in a total amount of less than 0.5% by weight based on the total weight of the feed. A preferred embodiment of the present invention relates to a method for inhibiting porcine epidemic diarrhea virus in animal feed, wherein benzoic acid is added to the animal feed in an amount of 0.25% to 0.475% by weight based on the total weight of the feed, and / or a powder comprising eugenol and thymol is added in an amount of 0.010% to 0.019% by weight based on the total weight of the feed. Commercially available benzoic acid tablets are relatively inexpensive compared to eugenol and thymol. The claimed solution is therefore cheaper than prior art solutions which merely comprise higher levels of essential oils.

[0009] The same low concentrations are also effective in mitigating Porcine Reproductive and Respiratory Syndrome virus (PRRSV) and Seneca Virus A (SVA). One embodiment of the present invention relates to a method for inhibiting PRRS virus and / or SVA in animal feed, wherein benzoic acid, eugenol and thymol are added to the animal feed in a total amount of less than 0.5% by weight based on the total weight of the feed. A preferred embodiment relates to a method for inhibiting PRRS virus and / or SVA in animal feed, wherein benzoic acid is added to the animal feed in an amount of 0.25% to 0.475% by weight based on the total weight of the feed, and / or a powder containing eugenol and thymol is added in an amount of 0.010% to 0.019% by weight based on the total weight of the feed.

[0010] Although the addition of benzoic acid, eugenol and thymol to the feed in a total amount of less than 0.5% by weight based on the total weight of the feed effectively mitigated all three viruses (i.e., PRRSV, SVA and PEDV), the mixture at this concentration acted faster on PEDV than on PRRSV and SVA. The rate of mitigation is very beneficial because it allows for a reduction in the holding time recommended by the Swine Health Information Center (SHIC) and other agencies. A preferred embodiment of the present invention relates to the use of a mixture comprising benzoic acid, eugenol and thymol for reducing the holding time required to be applied to allow degradation of porcine epidemic diarrhea virus in animal feed.

[0011] PCR (polymerase chain reaction) testing has been shown to be sensitive to PRRSV and PEDV. One embodiment of the present invention relates to a computer-implemented method of preparing a feed, the method comprising the steps of:

[0012] a) providing a sample of at least one feed ingredient and determining the Ct value of at least one predetermined virus by real-time quantitative reverse transcription polymerase chain reaction;

[0013] b) providing a model that defines a Ct value within a range in case the feed ingredient is contaminated with a virus;

[0014] c) using the model to determine whether a mitigating agent for inactivating viruses needs to be added to the feed comprising the feed ingredients of step a).

[0015] The Ct value represents the cycle fraction at which the instrument can first detect fluorescence from the amplification reaction (threshold cycle), as further explained, for example, in Bustin, SA, & Mueller, R. (2005), "Real-time reverse transcription PCR (qRT-PCR) and its potential use in clinical diagnosis" Clinical Science, 109 (4), 365-379. If the Ct value is "within range", the amount of virus is high enough to be easily detected by real-time quantitative reverse transcription polymerase chain reaction. If the Ct value is "out of range", the amount of virus is zero or so low that the virus will not cause any harm. For the viruses discussed herein, a Ct value ≥ 38 is an indication that the corresponding virus is not present. A Ct value ≤ 37 is an indication that the corresponding virus is present. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Different time spans are shown. DETAILED DESCRIPTION

[0017] The present invention relates in particular to a computer-implemented method for determining the need to add a mitigating agent to a feed to inactivate viruses that may be present in the feed ingredients intended to prepare the feed. A preferred mitigating agent is a mixture comprising benzoic acid tablets and a powder comprising eugenol and thymol. The mixture rapidly inactivates viruses even when added to the feed only in low concentrations. This is particularly applicable to (but not limited to) porcine epidemic diarrhea virus (PEDV). Thus, by the present invention, animal feed is rendered free of harmful viral RNA.

[0018] Animal feed of the present invention

[0019] The animal feed of the present invention can preferably be a feed for birds, fish or mammals. In a preferred embodiment of the present invention, the animal feed is a pig feed. Typical ingredients of pig feed are ground corn or other grains, and soybeans or other plant-based protein products.

[0020] The animal feed of the present invention comprises benzoic acid, eugenol and thymol, so that it is preferably free of viral RNA (ribonucleic acid). Preferably, "free of viral RNA" means that the corresponding Ct value is 38 or higher. More preferably, this means that no viral RNA can be detected by real-time quantitative reverse transcription polymerase chain reaction.

[0021] The present invention preferably relates to a virus selected from the group consisting of: porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus and Seneca virus. As used in the context of the present invention, "porcine epidemic diarrhea virus", PED virus and PEDV are synonyms. As used in the context of the present invention, porcine reproductive and respiratory syndrome virus, PRRS virus and PRRSV are synonyms. As used in the context of the present invention, Seneca virus A and SVA are synonyms. Therefore, "free of viral RNA" preferably means that no viral RNA from PEDV, PRRS and / or SVA can be detected by real-time quantitative reverse transcription polymerase chain reaction. When multiple viral strains are present, all strains are included. In the case of PRRSV, the preferred strain is PRRSV 174. This PRRSV strain is quite common in some pig-raising areas of the United States; it is considered to be highly toxic and serious.

[0022] In some embodiments, the term "viral RNA" preferably does not refer to viral RNA from Senecavirus A, because in the case of SVA, PCR may give false positive results. In this embodiment, "free of viral RNA" preferably means that no viral RNA from PEDV and PRRS can be detected by real-time quantitative reverse transcription polymerase chain reaction.

[0023] The animal feed of the present invention comprises benzoic acid, eugenol and thymol in an amount of preferably less than 0.5 wt%, more preferably less than 0.4 wt%, based on the total weight of the feed. In the most preferred embodiment, the animal feed of the present invention comprises benzoic acid tablets and powder, the powder comprising eugenol and thymol, wherein the total amount of benzoic acid tablets and the powder is 0.312 wt%, based on the total weight of the feed.

[0024] In another embodiment, the animal feed of the present invention comprises:

[0025] - 0.25% to 0.475% by weight, preferably 0.25% to 0.4% by weight, most preferably 0.25% to 0.39% by weight of benzoic acid, based on the total weight of the feed, and

[0026] - 0.010% to 0.019% by weight of a powder, based on the total weight of the feed, wherein the powder comprises eugenol and thymol, and

[0027] wherein the weight ratio between benzoic acid and eugenol is preferably at least 500:1, more preferably at least 833:1 and preferably at most 2500:1, and / or wherein the weight ratio between benzoic acid and thymol is preferably at least 250:1, more preferably at least 333:1 and preferably at most 500:1.

[0028] In one embodiment, the animal feed of the present invention comprises benzoic acid and a powder, wherein the powder comprises eugenol and thymol and preferably at least one auxiliary compound. Such a powder is Nutritional Products (Switzerland). In a preferred embodiment, the animal feed of the present invention comprises benzoic acid tablets and powder, wherein the powder comprises eugenol, thymol and at least one auxiliary compound, and wherein the powder comprises, based on the total weight of the powder, preferably 10% to 60% by weight, more preferably 10% to 53% by weight, and most preferably 10% to 30% by weight of the auxiliary compound. Eugenol and thymol are preferably in physical contact with the feed ingredients and any viruses that may be present. Therefore, the powder comprising eugenol, thymol and at least one auxiliary compound is preferably not coated. Therefore, the powder comprising eugenol, thymol and at least one auxiliary compound preferably contains less than 5% by weight of fully hydrogenated fats, partially hydrogenated fats and / or unhydrogenated fats. In these embodiments, it is preferred to add benzoic acid in an amount of 0.25% to 0.475% by weight, based on the total weight of the feed. This powder is preferably added in a total amount of 0.010% to 0.019% by weight, based on the total weight of the feed. Benzoic acid tablets are Commercially available from ELISA® Nutritional Products (Switzerland).

[0029] Method of the present invention

[0030] The present invention also relates to a method for inhibiting viruses in animal feed, wherein benzoic acid (preferably benzoic acid tablets) and a powder comprising eugenol and thymol are added to the animal feed in a total amount preferably less than 0.5% by weight, more preferably less than 0.48% by weight, based on the total weight of the feed. This method is suitable for inhibiting any kind of potentially harmful viruses. In a preferred embodiment, the present invention relates to a method for inhibiting porcine epidemic diarrhea virus, inhibiting porcine reproductive and respiratory syndrome virus, or inhibiting non-enveloped RNA viruses such as Seneca virus A. In the most preferred embodiment, the present invention relates to a method for inhibiting porcine epidemic diarrhea virus. In the method, preferably 0.25% to 0.475% by weight of benzoic acid based on the total weight of the feed is added to the feed, and the powder comprising eugenol and thymol is preferably added in an amount of 0.010% to 0.019% by weight based on the total weight of the feed.

[0031] In animal feed, the combination of benzoic acid, eugenol and thymol is preferably in physical contact with feed ingredients and any viruses that may be present. Therefore, the powder comprising eugenol and thymol should preferably not be coated with lipids. The powder comprising eugenol and thymol is preferably not a pellet. The powder comprising eugenol and thymol as used in the inventive method comprises based on the total weight of the powder, preferably less than 5 weight %, more preferably less than 4 weight %, even more preferably less than 3 weight % of fully hydrogenated, partially hydrogenated and / or unhydrogenated fat. Other auxiliary compounds such as fillers and glidants are acceptable. The powder comprising eugenol and thymol as used in the inventive method comprises based on the total weight of the powder, preferably 1 weight % to 50 weight %, more preferably 5 weight % to 40 weight %, most preferably 10 weight % to 30 weight % of auxiliary compounds.

[0032] Uses of the present invention

[0033] Most viruses have a half-life. Therefore, the half-life is the time it takes for half of a given amount of the virus to die naturally. Because the ability of viruses to survive in feed is limited, holding time is a way to mitigate the risk of viruses. Recently, the Swine Health Information Center (SHIC) has published guidelines on its website for calculating appropriate holding times.

[0034] In the context of the present invention, the term "holding time" refers to the period of time that a feed or feed ingredient is stored before being fed to an animal. Typical holding times are several days or even weeks.

[0035] A mixture comprising benzoic acid, eugenol and thymol inactivates RNA viruses in feed and feed ingredients. Thus, the holding time becomes moot, or at least greatly shortened. The present invention relates to the use of a mixture comprising benzoic acid, eugenol and thymol for reducing the holding time required upon application to allow degradation of viruses in animal feed. Thus, the holding time is preferably reduced to less than 7 days, more preferably less than 6 days, even more preferably less than 5 days, most preferably less than 3 days. In a preferred embodiment, viral RNA from porcine epidemic diarrhea virus, from porcine reproductive and respiratory syndrome virus and / or from Senecavirus A is degraded.

[0036] Thus, the present invention relates to the use of a mixture comprising benzoic acid, eugenol and thymol for reducing the holding time required upon application to allow degradation of viral RNA from porcine epidemic diarrhea virus, from porcine reproductive and respiratory syndrome virus and / or from Seneca virus A in animal feed, wherein the holding time is preferably reduced to less than 7 days, more preferably less than 6 days, even more preferably less than 5 days, most preferably less than 3 days. The most preferred embodiment relates to the use of a mixture comprising benzoic acid, eugenol and thymol for reducing the holding time required upon application to allow degradation of viral RNA from porcine epidemic diarrhea virus in animal feed, wherein the holding time is reduced to preferably less than 3 days.

[0037] In the mixtures relating to these embodiments, the weight ratio between benzoic acid and eugenol is preferably at least 500:1, more preferably at least 833:1 and preferably at most 2500:1, wherein the weight ratio between benzoic acid and thymol is preferably at least 250:1, more preferably at least 333:1 and preferably at most 500:1.

[0038] Computer-implemented method of the present invention

[0039] Typically, feed is prepared by a feed mill. For this purpose, the feed mill purchases the main feed ingredients (e.g. ground corn and soybean meal) and other feed ingredients (e.g. minerals, vitamins and other feed additives). A feed ingredient that is present in an amount of preferably at least 20% by weight, based on the total weight of the feed, is a "main feed ingredient".

[0040] Commercially available feed ingredients may become contaminated with harmful viruses, especially during their transportation or physical movement. Therefore, feed mills must take measures to ensure that the feed they sell does not contain harmful viruses. One measure is to store feed ingredients and / or feed for several days or weeks (holding time). This measure assumes that there is little or no risk of further contamination during the subsequent final movement of the feed to where it will be consumed.

[0041] The computer implemented method of the present invention allows the holding time to be reduced and / or made completely meaningless. In a preferred embodiment, the computer implemented method of the present invention ensures that a mitigating agent for inactivating the virus is added only if the presence of a harmful virus has been confirmed.

[0042] The computer-implemented method of the present invention can be based on various forms of hardware, software, firmware, processors, distributed servers (e.g., as used in cloud computing), or combinations thereof. In one embodiment, the computer-implemented method of the present invention involves the use of a communication infrastructure, such as the Internet.

[0043] The computer-implemented method of the present invention needs to determine the Ct value by real-time quantitative reverse transcription polymerase chain reaction. For this reason, a PCR device is required. Therefore, when implementing the computer-implemented method of the present invention, a combination of hardware and software is preferably used.

[0044] The software may be implemented as an application program tangibly embodied on a program storage device, or as different portions of the software implemented in the user's computing environment (e.g., as an applet) and in the reviewer's computing environment, where the reviewer may be located at a remote site (e.g., at a service provider's facility).

[0045] The Ct values ​​determined by real-time quantitative reverse transcription polymerase chain reaction are then used in a model that defines Ct values ​​within a range in the case of contamination of feed ingredients with viruses such as PEDV and PRRSV. In a preferred embodiment, the model of the invention is a decision tree. A decision tree is a model containing conditional control statements. In a particularly preferred embodiment, the method of the invention comprises at least one of the following conditional control statements:

[0046] If the Ct value of the determined PEDV is 37 or less,

[0047] It is recommended to add at least one demulcent (preferably a mixture of benzoic acid, eugenol and thymol) to feed containing feed ingredients whose Ct values ​​have been determined to be 37 or less,

[0048] Otherwise it is recommended to avoid adding mildew agents for inactivating the virus

[0049] If the Ct value of the determined PRRS is 37 or less,

[0050] It is recommended to add at least one demulcent (preferably a mixture of benzoic acid, eugenol and thymol) to feed containing feed ingredients whose Ct values ​​have been determined to be 37 or less,

[0051] Otherwise it is recommended to avoid adding mildew agents for inactivating the virus

[0052] If the Ct value of the non-enveloped virus (e.g., SVA virus) is 37 or less,

[0053] It is recommended to add at least one demulcent (preferably a mixture of benzoic acid, eugenol and thymol) to feed containing feed ingredients whose Ct values ​​have been determined to be 37 or less,

[0054] Otherwise it is recommended to avoid adding mildew agents for inactivating the virus

[0055] A Ct value of ≤37 indicates the presence of the corresponding virus, i.e., when the Ct value is ≤37, a mitigating agent for inactivating the virus should be added. In the most preferred embodiment, the method of the present invention comprises or consists of the following conditional control statements:

[0056] If the Ct value of the determined PEDV is 37 or less,

[0057] It is recommended to add at least one demulcent (preferably a mixture of benzoic acid, eugenol and thymol) to feed containing feed ingredients whose Ct values ​​have been determined to be 37 or less,

[0058] Otherwise it is recommended to avoid adding mildew agents for inactivating the virus

[0059] If the Ct value of the determined PRRS is 37 or less,

[0060] It is recommended to add at least one demulcent (preferably a mixture of benzoic acid, eugenol and thymol) to feed containing feed ingredients whose Ct values ​​have been determined to be 37 or less,

[0061] Otherwise it is recommended to avoid adding mildew agents for inactivating the virus

[0062] In one embodiment, an application for performing the method of the present invention is uploaded to and / or executed by a computer platform having hardware, such as one or more central processing units (CPU), random access memory (RAM), and input / output (I / O) interfaces. This typically includes at least one computer processor. The computer processor can be a processor typically found in a personal desktop computer, a portable computer, a mainframe, a minicomputer, a portable electronic device (e.g., a tablet computer or a smart phone). Various other peripherals, such as additional data storage devices and printing devices, can be connected to the computer platform. One embodiment of the present invention relates to a computer-implemented method for preparing a feed, the method comprising the following steps:

[0063] a) providing a sample of at least one feed ingredient and determining the Ct value of at least one predetermined virus by real-time quantitative reverse transcription polymerase chain reaction;

[0064] b) providing a model that defines a Ct value within a range in case the feed ingredient is contaminated with a virus;

[0065] c) using the model to determine whether a mitigating agent for inactivating viruses needs to be added to a feed comprising the feed ingredients of step a),

[0066] wherein the at least one feed ingredient is preferably a primary feed ingredient, and / or wherein the at least one feed ingredient is preferably selected from the group consisting of corn, ground corn, soy products, soy meal, canola meal, wheat and barley, and

[0067] Wherein the demulcent is preferably a mixture comprising benzoic acid, eugenol and thymol, and wherein if the determined Ct value of PEDV and / or PRRS is 37 or below, the mixture is added in an amount, preferably less than 0.5 wt %, based on the total weight of the feed.

[0068] Although the preferred moderators herein effectively inactivate non-enveloped viruses such as SVA, the moderators do not always completely decompose the RNA of non-enveloped viruses. In the case of inactivation but not complete decomposition of the virus, harmless RNA portions of non-viable viruses may remain. In such cases, the PCR test may provide a false positive result, and therefore, there is no benefit in determining the corresponding Ct value in an expensive and potentially time-consuming laboratory test. Therefore, one embodiment of the present invention relates to a computer-implemented method for determining whether a moderator needs to be added to a feed to inactivate viruses that may be present in a feed ingredient intended to prepare the feed, the method comprising:

[0069] a) providing a sample of at least one feed ingredient and determining the Ct value of at least one predetermined virus by real-time quantitative reverse transcription polymerase chain reaction;

[0070] b) providing a model that defines a Ct value within a range in case the feed ingredient is contaminated with a virus;

[0071] c) using the model to determine whether a demulcent (e.g. a mixture of benzoic acid, eugenol and thymol) needs to be added to a feed comprising the feed ingredients of step a),

[0072] Therein, a Ct value is not determined for Senecavirus A, and wherein preferably a Ct value is not determined for any non-enveloped virus.

[0073] Another embodiment of the present invention is directed to a computer-implemented method of preparing a feed, the method comprising the steps of:

[0074] a) providing a sample of at least one feed ingredient and determining the Ct value of at least one predetermined virus by real-time quantitative reverse transcription polymerase chain reaction;

[0075] b) providing a model that defines a Ct value within a range in case the feed ingredient is contaminated with a virus;

[0076] c) using the model to determine whether a mitigating agent for inactivating viruses needs to be added to a feed comprising the feed ingredients of step a),

[0077] wherein a Ct value is not determined for Senecavirus A, and wherein preferably a Ct value is not determined for any non-enveloped virus, and

[0078] Wherein steps b) and c) preferably include at least one of the following two actions:

[0079] Action 1:

[0080] If the Ct value of the determined PEDV is 37 or less,

[0081] then at least one moderator is added to a feed containing a feed ingredient having a Ct value determined to be 37 or less,

[0082] Otherwise avoid adding mildew agents used to inactivate the virus,

[0083] Action 2:

[0084] If the Ct value of the determined PRRS is 37 or less,

[0085] then at least one moderator is added to a feed containing a feed ingredient having a Ct value determined to be 37 or less,

[0086] Otherwise avoid adding mildew agents to inactivate the virus

[0087] Example

[0088] Example 1

[0089] In Example 1, an RNA virus was introduced into pig feed in a controlled manner.

[0090] A complete swine feed was prepared by mixing ground corn, soybean meal and other feed ingredients. The exact composition of the feed prepared in Example 1 is indicated in Table 1 below. A 2722 kg (2.722 metric ton) batch of feed was prepared in this manner.

[0091] Table 1

[0092]

[0093]

[0094] After the feed was manufactured, it was transported and placed in a farm feed storage bin. This feed was enough for 100 pigs for a period of 15 days. Once the feed bin had been filled, 454 grams of ice cubes that had been pre-frozen, stored at -80°C and containing the three selected RNA viruses were dropped into the top of the feed bin on day 0. Because the ice cubes fell on top of the batch of feed, they were allowed to melt naturally into liquid, allowing the three viruses to infiltrate the feed. Over the duration of the study (15 days), the infiltrated feed containing the three RNA viruses flowed to the bottom of the feed bin (by gravity) and was transferred to the piggery via a plastic tube containing a flexible auger. As the feed was gradually transferred to the piggery via the auger, some additional mixing occurred to provide the pigs with free access to the feed.

[0095] The three RNA viruses contained in the ice cubes are: Porcine reproductive and respiratory syndrome virus (PRRSV 174), porcine epidemic diarrhea virus (PEDV), and Seneca virus A (SVA). Of these viruses, SVA is the only non-enveloped virus. PRRSV 174 and PEDV are enveloped viruses. SVA is sometimes chosen as an alternative to foot-and-mouth disease virus (FMDV). FMDV is also a non-enveloped virus. The survival of enveloped viruses is generally different from the survival of non-enveloped viruses.

[0096] The composition of 454g / 454mL ice cubes is as follows:

[0097] 100 mL SVA (5 log TCID50 / mL, Ct=20.72), and

[0098] 100 mL PRRSV 174 (5 log TCID50 / mL, Ct=21.38), and

[0099] ·100mL PEDV (5log TCID50 / mL, Ct=24.25),

[0100] Balance with 154 mL of serum-free minimum essential media (MEM).

[0101] The Ct values ​​shown are related to PCR (polymerase chain reaction). It is generally believed that a Ct value of ≥ 38 is an indication of the absence of the corresponding virus. Therefore, the measured Ct values ​​from 20.72 to 24.5 confirm the presence of the corresponding virus.

[0102] Because the batch of feed delivered on Day 0 was expected to last for the duration of the study (15 days), a second block of ice (the same as used on Day 0) containing the three viruses was dropped on top of the remaining feed in the feed bin on Day 6. This was done to ensure that when the second block of ice melted, the remaining feed was also permeated with the three viruses and the feed gradually entered the pig house by gravity flow into the contained auger system which provided ad libitum feeding to the pigs until Day 15.

[0103] On day 6, a separate swiffer cloth was pulled over each of the six feed troughs in the pig house, in contact with feed particles present where the pigs were eating feed. Afterwards, the cloth was dipped into sterile saline and a 3 mL aliquot was poured out for testing. The samples were evaluated for the presence of viral nucleic acid by PCR.

[0104] On day 15, a separate swiffer cloth was tested again as described above. The results of the two PCR analyses are shown in Table 2. The symbol "5 / 6" means that 6 separate samples were tested from 6 feed troughs in the pig house, and 5 of the 6 feed trough samples (i.e., feeder samples) contained RNA of the corresponding virus. The symbols "6 / 6", "4 / 6" and "3 / 6" should be understood in the same way.

[0105] Table 2

[0106]

[0107]

[0108] dynamics:

[0109] When ice cubes fall on the top of the feed in the feed box, the ice cubes gradually and naturally melt, thereby allowing liquid to begin to penetrate downward into the feed from the upper part of the feed. Typical feed storage boxes, such as the feed storage boxes used in this study, have a conical bottom so that the stored feed can naturally flow downward into the hopper, where it is transferred to the barn by a flexible auger, and deposited into any number of automatic feeders with storage hoppers positioned in the piggery. When the pigs raised in the piggery need the feed in the external storage box, the feed is pumped into the auger from the bottom center of the box. In general, feed is extracted faster from the center of the box, and the feed closest to the outer periphery of the box tends to flow more slowly and enter the piggery later. Therefore, when ice cubes fall on the top of the feed in the center of the box and begin to melt, the infiltrated feed flows into the auger and the piggery faster than the rest of the feed. If the ice cubes are not completely melted when they arrive at the auger, the melting and dispersion of the ice cubes are further completed by the mechanical action of the auger transferring the infiltrated feed into the piggery. Thus, when the feed is delivered to the pig house, the mechanical action of the auger completes further mixing and penetration. An independent auger passes through the wall of the pig house (sealed around the auger) to deliver the feed to the pigs. During the winter (in Minnesota, USA), when ice melts more slowly in the feed box, the higher temperature sustained by the pigs in the pig house ensures that when the feed passes through the auger to the automatic feeder, any remaining ice particles in the feed become completely melted, so that the liquid has completely penetrated into the feed before being consumed by the pigs. Infiltrating the feed with the virus at lower temperatures increases the possibility that the virus remains infectious for a longer period of time. After 6 days, a second ice cube is dropped on top of the remaining feed in the box, ensuring that the feed consumed in the next 9 days (until the 15th day) is infiltrated in the same way as the feed during the first 6 days.

[0110] Interpretation of the data shown in Table 2:

[0111] Checking the feeders for the presence of viral RNA on days 6 and 15, respectively, is a test of whether the expected viral RNA was successfully introduced into the feed and feed delivery system, and whether it is viable to be ingested by the pigs. Different results were obtained on days 6 and 15 for all three viruses. Apparently, it was difficult to thoroughly contaminate the feed entering the barn in the first 6 days by simply dropping a single ice cube into the center of the box on day 0, but the feed became more thoroughly contaminated during the remaining 9 days after the second ice cube was added on day 6. The results in Table 2 also show that detecting non-enveloped SVA is challenging. However, the presence of SVA in this batch of feed was unquestionable, because on day 15, all pigs showed clinical signs of SVA, regardless of whether they were fed from the feed trough / pen in the barn.

[0112] Example 2

[0113] In Example 2, the test of Example 1 was repeated at the same time. However, in Example 2, a mixture containing benzoic acid tablets and a powder containing thymol and eugenol was added, and the feed composition shown in Table 1 was included. A feed containing 0.312 wt% of the mixture instead of an equal amount of ground corn based on the total weight of the feed was prepared.

[0114] Feeder samples were then taken on day 6 (i.e., 6 days after the introduction of the first ice cube into the feed) and day 15 (i.e., 9 days after the introduction of the second ice cube) as explained in Example 1. The different time spans, "day 0 → day 6" and "day 6 → day 15", were Figure 1 The results are shown in Table 3 below.

[0115] The results in Table 3 show that a mixture comprising benzoic acid tablets and a powder comprising thymol and eugenol (total 0.312 wt % based on total feed weight) is a very effective way to reduce the presence of three different types of viruses in feed, namely PRRSV, PEDV and SVA. In the case of PRRSV and SVA, the treatment was more effective if the viruses had been exposed to benzoic acid, thymol and eugenol for a longer period of time (9 days after the initial 6 days). In the case of PEDV, there was no additional benefit to increasing the exposure time. In other words, the mixture of benzoic acid, thymol and eugenol quickly inactivated PEDV.

[0116] Rapid inactivation of viruses is beneficial because pig farmers prefer not to hold or isolate feed for storage until the animals need it. The need for any holding time, especially longer times, makes the farmer's life more complicated and increases the costs associated with storage and logistics for efficient pork production. In addition, longer holding times can reduce the quality of the feed because the stability and retention of some essential nutrients (i.e., vitamins) decrease over longer periods of time. On the other hand, farmers are very willing to accept and use feed holding times if they will reduce the risk of animal disease transmission, which may cause illness to the farmer's animals and / or increase the possibility of greater animal and economic losses within the entire pork industry.

[0117] Table 3

[0118]

[0119] * ≥9 days may be any exposure from 9 to 15 days; see subsection “Kinetics” above

[0120] Example 3

[0121] In Example 3, the effect of different concentrations of the mixture of Example 2 (i.e., benzoic acid, thymol, and eugenol) in the feed on the detection of three viral RNAs in feeder samples on day 6 is described. The two concentrations tested were:

[0122] Low concentration: 0.312 wt% of the mixture of Example 2 based on the total weight of the feed.

[0123] High concentration: 0.52 wt% of the mixture of Example 2 based on the total weight of the feed.

[0124] In Example 3, the test of Example 2 was repeated simultaneously. For details of the test method, see Example 2.

[0125] On day 6, feeder samples were taken and analyzed as described in the previous examples. The results are shown in Table 4, which also includes the results from Examples 1 and 2.

[0126] Table 4

[0127]

[0128] The results show that the mixture of benzoic acid, thymol and eugenol is already effective against PRRSV, PEDV and SVA at the lower of the two concentrations. For these viruses, there is no need to increase the concentration of the mixture from 0.312 wt % to 0.52 wt % based on the total weight of the feed. This is highly relevant to farmers, as any need for higher amounts of feed additives will increase costs.

[0129] Example 4

[0130] The summary of the results of Examples 2 and 3 shows that the mixture of benzoic acid, thymol and eugenol has outstanding performance against PEDV. More specifically, these examples show that the mixture of benzoic acid, thymol and eugenol is effective against PEDV (i) after a short period of time and (ii) at low concentrations.

[0131] In some of the feed samples tested, RNA from PEDV could be detected by PCR, even though a mixture of benzoic acid, thymol and eugenol had been added to the corresponding feed. However, a positive RNA result does not always indicate the presence of live virus. In the case of inactivated virus, it is also possible to obtain a positive RNA result in the PCR test (i.e. a false positive result in the PCR test). False positive results in the PCR test can be detected by looking at the corresponding animal data: the animals would not become ill from ingesting non-live virus.

[0132] To demonstrate that 0.312 wt% of the mixture based on the total weight of the feed was sufficient to inactivate PEDV, the following was performed:

[0133] Measure the average growth rate of pigs from day 0 to day 15 (average daily gain, ADG)

[0134] Pigs were scored for the presence of clinical signs (diarrhea as evidence of disease) on day 15

[0135] Testing pigs for PEDV in rectal swab samples collected on day 15 (as evidence of infection)

[0136] The results in Table 5 below show that any PEDV RNA detected by PCR in feeder samples taken on days 6 and 15 was derived from inactivated, and therefore harmless, PEDV.

[0137] Table 5

[0138]

[0139] Table 6 also demonstrates that if ice cubes containing PEDV are melted in feed without benzoic acid, thymol and eugenol, pigs will be infected with PEDV. In addition, Table 6 shows that lower concentrations of the mixture according to the invention are sufficient for inactivating PEDV in feed.

[0140] Example 5

[0141] In Example 5, the effects of PRRSV and SVA on pigs that had consumed a feed with or without the mixture were determined as explained in Example 4. Table 6 shows the complete data, including data from Example 4:

[0142] Table 6

[0143]

[0144] 1 Represents the percentage of animals with the virus recovered from biological specimens (PRRSV = serum, PEDV = rectal swab, or SVA = tonsil).

[0145] 2 The percentage of pens showing clinical signs of disease (dyspnea / weight loss / coarse coat = PRRSV, diarrhea = PEDV, or lameness / blistering = SVA) is indicated.

Claims

1. Non-therapeutic use of a mixture comprising benzoic acid, eugenol and thymol for reducing the holding time required to allow viral degradation in animal feed or in feed ingredients applied, wherein viral RNA from porcine reproductive and respiratory syndrome virus and / or from Seneca virus A is degraded during the shortened holding time.

2. The use according to claim 1, wherein the holding time is reduced to less than 7 days.

3. The use according to claim 1 or 2, wherein the weight ratio between benzoic acid and eugenol is at least 500:1, and / or The weight ratio between benzoic acid and thymol is at least 250:

1.

4. The use according to claim 1 or 2, wherein 0.25 wt% to 0.475 wt% of benzoic acid tablets are added based on the total weight of the feed, and / or The powder comprising eugenol and thymol is added in an amount of 0.010 wt % to 0.019 wt % based on the total weight of the feed.

5. The use according to claim 4, wherein the powder comprises from 10% to 60% by weight of auxiliary compounds, based on the total weight of the powder, and / or wherein the powder comprises less than 5% by weight of fully hydrogenated fats, partially hydrogenated fats or non-hydrogenated fats.

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