Detection of antibiotic and / or chemical use in animals using epigenetic methods

Abstract

The present invention relates to a method of determining whether a test animal and / or a product-obtaining test animal has received and / or is currently receiving treatment with at least one antibiotic and / or veterinary chemical, comprising: (a) determining a test methylation profile from genomic material, the genomic material is contained in a biological sample obtained from a test animal and / or an animal-derived product; and (b) comparing the test methylation profile obtained from (a) to a reference methylation profile obtained from a control animal of the same biometric population of the test animal wherein the control animal has not been accepted and / or has not currently accepted treatment with at least one antibiotic and / or veterinary chemical, wherein a difference in the test methylation profile of (a) compared to a reference methylation profile from a control animal indicates that the test animal has accepted and / or is currently accepted treatment with at least one antibiotic and / or veterinary chemical; and wherein the test animal is a terrestrial animal, and wherein the veterinary chemical is an antiparasitic agent, an antiviral agent, a feed additive, a water additive, a disinfectant, glutaraldehyde and / or formalin.

Description

Technical Field

[0001] The present invention relates to a method for detecting at least one antibiotic and / or veterinary chemical used and / or applied to an animal using an epigenetic method. Specifically, the method can detect the use of at least one antibiotic or the application of at least one antibiotic and / or veterinary chemical in the breeding or feeding process of at least one terrestrial animal by determining the methylation state of CpG sites in the test animal and comparing the obtained methylation state with the reference methylation state of a control animal bred without the use or application of the antibiotic and / or veterinary chemical. Background Art

[0002] Antibiotics are used in human and veterinary medicine to treat bacterial diseases. Antibiotics are also used prophylactically at low levels in feed or water to improve growth rates and mortality levels in livestock. However, overuse of antibiotics, especially in agriculture, has been associated with an increase in antibiotic-resistant bacteria, which will ultimately compromise the ability to treat bacterial diseases in humans and animals alike. In addition, antibiotic use has been associated with other unexpected and adverse physiological consequences, such as low birth weight and increased susceptibility to disease in infants born to mothers treated with antibiotics. Therefore, tools for assessing not only current antibiotic use but also historical antibiotic use are highly attractive in livestock production for the purpose of guiding breeding, feeding, veterinary and / or management practices. In addition, analysis of meat and meat products to assess historical antibiotic use of individual animals prior to slaughter can be highly attractive for the auditing, certification and labeling of meat products.

[0003] Until now, antibiotic testing has involved chemical assays to find antibiotic residues present in the blood or tissues of animals. However, most antibiotics used to promote growth are either added to feed or added to water, and most antibiotics in these classes are poorly absorbed in the gastrointestinal system, so residue testing in blood or tissues may not capture use of all antibiotic classes, especially after a withdrawal period (in which animals are no longer given antibiotics) has begun. There is also no way to test animals or meat products for prior antibiotic use unless the antibiotic residues are in the blood or tissues.

[0004] Therefore, there is a need in the art for a simple and accurate method to detect current and past antibiotic and / or drug use in animals, particularly livestock.

[0005] Epigenetics is the study of heritable traits that result from mechanisms other than changes in the underlying DNA sequence. In other words, epigenetic marks "program" our genes. Epigenetic marks can be chemical marks (such as methylation), protein-based marks (such as histones), or a combination of both. During development and cell differentiation, DNA methylation is dynamic, but some DNA methylation patterns can be retained, accumulated, and / or inherited to the next generation as a form of epigenetic memory. These changes may be the cause of heritable changes in gene activity, as DNA methylation events have been shown to be a regulatory mechanism associated with gene silencing, expression, chromatin remodeling, or imprinting. Epigenetics is attractive for animal breeding because it can identify the causality and heritability of complex traits and diseases. DNA methylation patterns are modified during an individual's lifetime by many environmental factors such as diet, stress, drugs, or pollution. Some environments are more likely to increase certain methylation patterns, and these patterns may lead to epigenetic and / or phenotypic differences between individuals.

[0006] Several studies have shown that antibiotic exposure can alter epigenetic marks in eukaryotic cells. For example, in a study investigating the relationship between antibiotic use during pregnancy and birth weight outcomes, specific regions of the genome were found to be differentially methylated in response to antibiotic exposure (Vidal et al., 2013). A study conducted on cultured plant cells observed changes in global DNA methylation when cells were exposed to the antibiotic kanamycin (Bardini et al., 2003). However, the art is silent on epigenetic changes that may occur as a result of current and / or past antibiotic and / or drug use in animals, particularly livestock.

[0007] Therefore, there remains a need in the art for a simple and accurate method to detect current and / or past antibiotic and / or drug use in animals, particularly livestock. Summary of the invention

[0008] The present invention solves the above problems by providing a method for developing a new type of analysis using changes in DNA methylation patterns to evaluate the use of antibiotics or other chemicals in terrestrial agricultural animal production, meat and / or meat products. Specifically, the method includes a method for detecting the use of antibiotics and / or veterinary chemicals in test animals and / or test animals from which products are derived, the method comparing the methylation state of at least one CpG site in the test animal with the corresponding CpG site in the control animal and / or the control animal from which the product is derived, wherein the control animal has not used antibiotics and / or veterinary chemicals, and wherein the presence of hypomethylation or hypermethylation at the CpG site in the test animal indicates that the test animal has been treated with antibiotics and / or veterinary chemicals. The method according to any aspect of the present invention is a simple and accurate method for detecting current and / or past antibiotic and / or drug use in animals.

[0009] The present invention is based on the discovery that contact of animals with antibiotics and / or veterinary chemicals leads to stress in the animal's internal environment, and that this can permanently change the animal's genome through epigenetics. Specifically, the ability to adapt to the environment (i.e. the presence of antibiotics and / or veterinary chemicals) and maintain the adapted biological pattern depends on epigenetic mechanisms, including DNA methylation. Specifically, the present invention is based on the discovery that contact with antibiotics and / or veterinary chemicals can also lead to changes in the animal's epigenetic mechanisms, including DNA methylation patterns, and that these patterns can also be transmitted to different products that may be derived from the animals.

[0010] The inventors have unexpectedly discovered that this property can be used to identify "epigenetic fingerprints" on the genome that are specific for the presence of antibiotics and / or veterinary chemicals in not only one animal, but potentially all animals that have been exposed to at least one antibiotic and / or veterinary chemical. For example, these "epigenetic fingerprints" may be specific not only to animals that have been exposed to antibiotics and / or veterinary chemicals, but also to any products derived from that animal (individual). Based on these findings, the present invention provides methods for detecting the use of antibiotics and / or veterinary chemicals in animals, particularly terrestrial animals, such as farmed animals also known as livestock and poultry, from which animal-derived products come. In this way, the method according to any aspect of the present invention can then be used to accurately and reliably determine whether any test animal or its product has been exposed to or treated with at least one antibiotic and / or veterinary chemical. The method according to any aspect of the present invention can then also use DNA methylation analysis to distinguish between the prophylactic use of antibiotics (growth promotion) and the therapeutic use of antibiotics. DNA methylation analysis can also be a step to clarify whether animals treated with veterinary chemicals and / or antibiotics are given a withdrawal period of the veterinary chemicals and / or antibiotics before slaughter, or to distinguish between the use of different classes of antibiotics. The methods according to the present invention can also be used to evaluate current and historical antibiotic use in livestock or meat to study epigenetic markers, which can then be used to create assays to test antibiotic use in terrestrial livestock species. In addition, the methods according to any aspect of the present invention can then be used to predict and avoid undesirable traits for animal welfare and for more sustainable livestock production that are contributed to by the use of antibiotics and / or veterinary chemicals. In one example, the methods according to any aspect of the present invention can also be used to distinguish between the routes of administration of antibiotics (i.e., water vs. feed vs. injection type).

[0011] According to one aspect of the present invention, there is provided a method for determining whether a test animal and / or a test animal from which a product is derived has been and / or is currently receiving treatment with at least one antibiotic and / or veterinary chemical, the method comprising:

[0012] (a) determining a test methylation profile from genomic material contained in a biological sample obtained from a test animal and / or an animal-derived product; and

[0013] (b) comparing the test methylation profile obtained from (a) with a reference methylation profile obtained from a control animal, wherein the control animal has not been and / or is not currently being treated with at least one antibiotic and / or veterinary chemical,

[0014] wherein a difference in the test methylation profile of (a) compared to a reference methylation profile from a control animal indicates that the test animal has received and / or is currently receiving treatment with at least one antibiotic and / or veterinary chemical; and

[0015] wherein the test animal is a terrestrial animal, and wherein the veterinary chemical is an antiparasitic drug, an antiviral drug, a feed additive, a water additive, a disinfectant, glutaraldehyde and / or formalin.

[0016] In one example, the veterinary chemical is at least one antimicrobial drug, such as an antiparasitic drug, an antiviral drug, or an antibiotic.

[0017] According to another aspect of the present invention, there is provided a method for determining whether a test animal and / or a test animal from which a product is derived has received and / or is currently receiving treatment with at least one antibiotic, the method comprising:

[0018] (a) determining a test methylation profile from genomic material contained in a biological sample obtained from a test animal and / or an animal-derived product; and

[0019] (b) comparing the test methylation profile obtained from (a) with a reference methylation profile obtained from a control animal, wherein the control animal has not been and / or is not currently being treated with at least one antibiotic,

[0020] Wherein a difference in the test methylation profile of (a) compared to the reference methylation profile from a control animal indicates that the test animal has received and / or is currently receiving treatment with at least one antibiotic.

[0021] According to a further aspect of the present invention, there is provided a method for determining whether a test animal and / or a test animal from which a product is derived has received and / or is currently receiving treatment with at least one antibiotic or veterinary chemical or a combination thereof, the method comprising:

[0022] (a) determining the methylation status of at least one CpG site in genomic material contained in a biological sample obtained from a test animal,

[0023] (b) comparing the methylation status of the CpG sites from (a) with the methylation status of a control animal that has not been and / or is not currently being treated with at least one antibiotic and / or veterinary chemical,

[0024] wherein a difference in the test methylation status of (a) in the test animal compared to the CpG site in the control animal indicates that the test animal has received and / or is currently receiving treatment with at least one antibiotic and / or veterinary chemical; and

[0025] wherein the test animal is a terrestrial animal, and wherein the veterinary chemical is an antiparasitic drug, an antiviral drug, a feed additive, a water additive, a disinfectant, glutaraldehyde and / or formalin.

[0026] In one example, the veterinary chemical is at least one antimicrobial drug, such as an antiparasitic drug, an antiviral drug, or an antibiotic.

[0027] The difference in methylation status according to any aspect of the invention is hypomethylation or hypermethylation of the CpG site in the test animal, and the hypomethylation and hypermethylation of the CpG site indicate that the test animal has received and / or is currently receiving treatment with at least one antibiotic and / or drug.

[0028] As used herein, the term "terrestrial animal" refers to any organism that lives entirely on land or lives primarily on land, particularly compared to aquatic animals. Specifically, the terrestrial animal according to any aspect of the present invention can be any animal in the animal kingdom. More specifically, the terrestrial animal according to any aspect of the present invention can be a mammal or a bird. Even more specifically, the terrestrial animal according to any aspect of the present invention can be a farmed animal selected from livestock or poultry. Specifically, livestock can include cattle, sheep, pigs, goats, horses, camels, donkeys, mules, rabbits, etc., and poultry can include chickens, turkeys and other Galliformes birds, ducks, geese, quails, etc. As used herein, the term "livestock" can also include poultry and refers to any farm animal or animal that can be used for agriculture. More specifically, the terrestrial animal according to any aspect of the present invention can be selected from cattle, sheep, pigs, goats, horses, camels, donkeys, mules, rabbits, chickens, turkeys, ducks, geese and quails.

[0029] As used herein, the term 'animal-derived product' refers to a product derived from an animal. Specifically, the term 'test animal-derived product' refers to the sample or object discussed that will be subjected to the method according to any aspect of the present invention. These products from animals may include meat and meat products, and also include fat, meat, blood, processed meat and rare products, such as fish glue and rennet, poultry products (meat and eggs), dairy products (milk and cheese) and non-food products such as fiber (wool, mohair, cashmere, leather, etc.). Animal-derived products may also include products that can be prepared using animal products (such as fat), such as soap, cream, etc. In an example, animal-derived products are meat, eggs, blood, brain, sperm, milk and any other tissue or sample providing genomic DNA. Specifically, animal-derived products are meat. In an example, animal-derived product samples can be a single type of meat, different types of meat, a single part of a type of meat, different parts of a single type of meat or different parts of different types of meat. The sample can be from any biological entity with a DNA genome and DNA genome methylation. Specifically, the methylation site is a CpG site.

[0030] In one example, the test animal and / or the test animal-derived product can be selected from fattening pigs, fattening cattle, piglets, fattening turkeys, fattening chickens, veal calf, horses, lambs, fattening rabbits, sheep dairy products, fattening horses, fattening lambs, fattening sheep, dairy cows, laying hens, piglet sows, goat dairy products, etc.

[0031] The term "test" used in conjunction with the term object and / or animal in the present disclosure refers to an entity subjected to the method according to any aspect of the present invention and is the basis of the analytical application of the present invention. Therefore, a "(individual) test object", "(individual) test object group" or "test profile" or "test animal-derived product" is an (individual) object or group of objects to be tested according to the present invention or a profile obtained or generated in this context. In contrast, the term "reference" or "control" should refer to an entity that is mostly predetermined for comparison with the test entity. For example, the term "reference animal" is used interchangeably with "control animal", which refers to an animal of the same biological taxonomic group for comparison or as a control for referring to a 'test animal'. Similarly, the term 'sample' and / or 'test animal-derived product sample' used in accordance with any aspect of the present invention refers to an entity that can be subjected to the method of the present invention. Specifically, a sample can be any (test) animal-derived product that can be subjected to the method of the present invention to determine whether the test animal from which the test animal-derived product is derived has been or has been exposed to at least one antibiotic and / or veterinary chemical. For example, the animal-derived product sample may be a piece of meat that is tested according to any aspect of the invention to determine whether the test animal from which the test animal-derived product was derived has been exposed to at least one antibiotic and / or veterinary chemical. In another example, the (test) animal-derived product sample may be milk that is tested according to any aspect of the invention to determine whether the test animal from which the test animal-derived product was derived is still currently exposed to at least one antibiotic and / or veterinary chemical. Blockchain may also be used to make information easily accessible to consumers.

[0032] As used herein, the term "comprising" should be interpreted as covering both "including" and "consisting of", both of which are clearly intended, and are therefore aspects of the individual disclosures of the present invention. When used herein, "and / or" should be considered to specifically disclose each of two specified features or components, with or without the other. For example, "A and / or B" should be considered to be the specific disclosure of (i) A, (ii) B, and (iii) A and B, respectively, as if each is individually set forth herein. In the context of the present invention, the terms "about" and "approximately" represent that those skilled in the art will understand that the accuracy interval of the technical effect of the feature in question is still ensured. The terms generally indicate deviations of ±20%, ±15%, ±10%, and, for example, ±5% from the indicated numerical value. As will be understood by a person of ordinary skill, the specific deviation of the numerical value of a given technical effect will depend on the nature of the technical effect. For example, natural or biotechnological effects can generally have such deviations that are greater than artificial or engineering technical effects. When an indefinite or definite article is used when referring to a singular noun, for example "a", "an" or "the", this includes a plural of that noun unless something else is specifically stated.

[0033] In the context of the present invention, the terms "methylation profile", "methylation pattern", "methylation status" or "methylation state" are used herein to describe the condition, situation or situation of methylation of a genomic sequence, and such terms refer to the characteristics of a DNA segment at a specific genomic locus that are associated with methylation. Such characteristics include, but are not limited to, whether any cytosine (C) residues within the DNA sequence are methylated, the location of methylated C residues, the percentage of methylated C at any particular residue segment, and allelic differences in methylation due to, for example, differences in allelic origin. DNA fragments may also include specific preselected methylation sites.

[0034] As used herein, the term "preselected methylation sites" refers to methylation sites selected from genes or regions that showed the highest degree of methylation changes during the training process of the method, and meet certain quality criteria, such as considering a minimum sequencing coverage of ≥5x and for ≥5 qualified CpG sites. In addition, genes with an average methylation level of <0.1 or an average methylation level of >0.9 can be excluded due to their limited dynamic range. A "reference methylation profile" can be defined based on multiple training samples using multivariate statistical methods such as principal component analysis or multidimensional scaling analysis.

[0035] The term "methylation state" refers to the state of a specific methylation site (i.e., methylation relative to non-methylation), which means that a residue or methylation site is methylated or unmethylated. Then, based on the methylation state of one or more methylation sites, a methylation profile can be determined. Accordingly, the term "methylation profile" or "methylation pattern" refers to the relative concentration or absolute concentration of methylated C residues or unmethylated C residues at any specific residue segment in the genomic material of a biological sample. For example, if a cytosine (C) residue that is not usually methylated in a DNA sequence is methylated, it can be referred to as "hypermethylated"; and if a cytosine (C) residue that is usually methylated in a DNA sequence is not methylated, it can be referred to as "hypomethylated". Similarly, if a cytosine (C) residue in a DNA sequence (e.g., DNA from a sample nucleic acid of a test subject) is methylated compared to another sequence from a different region or from a different individual (e.g., relative to a standard nucleic acid of a normal nucleic acid or a reference sequence), the sequence is considered to be hypermethylated compared to another sequence. Alternatively, if the cytosine (C) residues within a DNA sequence are non-methylated compared to another sequence from a different region or from a different individual, the sequence is considered to be hypomethylated compared to the other sequence. These sequences are referred to as "differentially methylated". The measurement of differential methylation levels can be accomplished in a variety of ways known to those skilled in the art. As a non-limiting example, one method is to measure the methylation levels of the CpG sites of each query determined by a bisulfite sequencing method.

[0036] The term "hypermethylation" refers to an average methylation status corresponding to an increased presence of 5-mCyt at one or more CpG dinucleotides within the DNA sequence of a test DNA sample relative to the amount of 5-mCyt found at the corresponding CpG dinucleotides within a normal control DNA sample. Specifically, a control refers to an animal-derived product or animal that has not been exposed to antibiotics and / or veterinary chemicals.

[0037] The term "hypomethylation" refers to an average methylation status corresponding to a reduced presence of 5-mCyt at one or more CpG dinucleotides within the DNA sequence of a test DNA sample relative to the amount of 5-mCyt found at the corresponding CpG dinucleotides within a normal control DNA sample. Specifically, a control refers to an animal-derived product or animal that has not been exposed to antibiotics and / or veterinary chemicals.

[0038] As used herein, "methylated nucleotide" or "methylated nucleotide base" refers to the presence of a methyl moiety on a nucleotide base, wherein the methyl moiety is not generally present in a recognized typical nucleotide base. For example, cytosine in its conventional form does not contain a methyl moiety on its pyrimidine ring, but 5-methylcytosine contains a methyl moiety at position 5 of its pyrimidine ring. Therefore, cytosine in its conventional form may not be considered a methylated nucleotide, while 5-methylcytosine may be considered a methylated nucleotide. In another example, thymine may contain a methyl moiety at position 5 of its pyrimidine ring, however, for the purposes of this article, thymine may not be considered a methylated nucleotide when present in DNA. Typical nucleotide bases for DNA are thymine, adenine, cytosine, and guanine. Typical bases for RNA are uracil, adenine, cytosine, and guanine. Accordingly, a "methylation site" is a location in a target gene nucleic acid region where methylation is possible. For example, a location containing CpG is a methylation site where cytosine may be methylated or may not be methylated. In particular, the term "methylated nucleotide" refers to a nucleotide carrying a methyl group attached to a nucleotide position where methylation can occur. These methylated nucleotides are commonly found in nature, and to date, methylated cytosine, which occurs mainly in the context of the dinucleotide CpG, but also in the context of CpNpG- and CpNpN-sequences, can be considered the most common. In principle, other naturally occurring nucleotides may also be methylated, but are not taken into account for any aspect of the present invention.

[0039] As used herein, "CpG sites" or "methylation sites" are nucleotides within nucleic acids (DNA or RNA) that are susceptible to methylation, either by naturally occurring events in vivo or by intentional chemical methylation of nucleotides in vitro. Some of these sites may be hypermethylated and some may be hypomethylated in animals exposed to antibiotics and / or veterinary chemicals compared to cells not exposed to antibiotics and / or veterinary chemicals.

[0040] As used herein, "CpG island" describes a DNA sequence that contains a functionally or structurally deviated CpG density. For example, Yamada et al. have described a set of criteria for determining a CpG island: it must be at least 400 nucleotides in length, must have a GC content greater than 50%, and must have an OCF / ECF ratio greater than 0.6 (Yamada et al., 2004, Genome Research, 14, 247-266). Others define CpG islands more broadly as sequences of at least 200 nucleotides in length, having a GC content greater than 50% and an OCF / ECF ratio greater than 0.6 (Takai et al., 2002, Proc. Natl. Acad. Sci. USA, 99, 3740-3745). In the context of the present invention, the terms "methylation profile", "methylation pattern", "methylation status" or "methylation state" are used herein to describe the condition, situation or situation of methylation of a genomic sequence, and such terms refer to characteristics of a DNA segment at a specific genomic locus that are associated with methylation. Such characteristics include, but are not limited to, whether any cytosine (C) residues within the DNA sequence are methylated, the location of methylated C residues, the percentage of methylated C at any specific residue stretch, and allelic differences in methylation due to, for example, differences in allelic origin.

[0041] As used herein, a "methylated nucleic acid molecule" refers to a nucleic acid molecule containing one or more nucleotides that are methylated.

[0042] As used herein, the term 'epigenetic changes' refers to chemical (e.g., methylation) changes or protein (e.g., histone) changes that occur in the body of a gene or its promoter. Through epigenetic changes, environmental factors (such as diet, stress, and prenatal nutrition) can produce genetic imprints that are passed from one generation to the next.

[0043] As used herein, the term "genomic material" refers to a nucleic acid molecule or genomic fragment of a subject or group of subjects. Specifically, such nucleic acid molecules or fragments are DNA or RNA or hybrids thereof, and most preferably are molecules of the DNA genome of the subject or group of subjects.

[0044] As used herein, "DNA sample" refers to DNA extracted from a terrestrial animal according to any aspect of the present invention using methods known in the art.

[0045] Specifically, when differential methylation is detected in a test animal, that is, the test animal and / or a product derived from the test animal shows hypermethylation or hypomethylation at at least one CpG site compared to a control group (i.e., an animal not exposed to at least one antibiotic and / or veterinary chemical), the test animal is or has been exposed to at least one antibiotic and / or veterinary chemical. The difference in methylation according to any aspect of the invention may be hypomethylation or hypermethylation.

[0046] A "biological sample" in the context of the present invention may comprise any biological material containing genomic material obtained from a subject or group of subjects, and may be liquid, solid or both, may be tissue or bone, or body fluids such as blood, lymph, etc. Specifically, the biological sample useful in the present invention may comprise biological cells or fragments thereof. More specifically, the biological sample is selected from blood, brain, sperm, milk and any other tissue or sample that provides genomic DNA.

[0047] The one or more preselected methylation sites in (a) are methylation sites associated with tissue-specific gene expression, preferably wherein the preselected methylation sites are associated with gene expression in a unique tissue.

[0048] The tissue may be selected from:

[0049] (i) metabolic tissue such as intestinal tissue, preferably the ileum or jejunum,

[0050] (ii) muscle tissue,

[0051] (iii) skin tissue, and

[0052] (iv) organ tissue, preferably liver and / or pancreatic tissue.

[0053] As used herein, the term "antibiotic" refers to any drug that can be administered to a terrestrial animal for therapeutic and / or preventive purposes. The antibiotic can be administered by any method known in the art. The antibiotic can be orally administered to a terrestrial animal according to any aspect of the present invention in the animal's feed or water so that it is ingested. In another example, the antibiotic can be injected into the animal. In one example, the antibiotic can be introduced into a terrestrial animal via mammary injection. A skilled artisan will understand the best method of providing the antibiotic to an animal based on the specific taxonomic group of the animal, the class of antibiotic, and the disease being treated or prevented. Specifically, the antibiotic according to any aspect of the present invention can be selected from the following categories: amide alcohols, aminocyclins, aminoglycosides, ansamycins, β-lactams, carbaephems, carbapenems, cephalosporins, chloramphenicol, fluoroquinolones, glycopeptides, glycylcyclines, ketolides, lincosamides, lipopeptides, macrolides, monocyclic β-lactams, nitrofurans, nitroimidazoles, oxazolidinones, penicillins, phosphonic acid derivatives, pleuromutilins, polymyxins, polypeptides, quinolones, rifamycins, riminofenazines, steroid antibiotics, streptogramins, sulfonamides, tetracyclines and trimethoprim. More specifically, the antibiotic can be selected from tetracycline hydrochloride, amoxicillin and colistin.

[0054] At least one or more of the above-mentioned antibiotics may be supplied simultaneously or continuously to the test animals according to any aspect of the present invention. Contact of antibiotics with terrestrial animals may bring about epigenetic changes, at least DNA methylation changes, which may then be determined using the methods according to any aspect of the present invention. The concentration of antibiotics in each dose that has been given to the test animals and / or the period of time during which the antibiotics are given may affect the degree of differential methylation of the test animals relative to the control animals. It is within the knowledge of the technician to determine the concentration of each dose required for the test animals and the period of antibiotic exposure, depending on whether the antibiotic is given for preventive or therapeutic measures.

[0055] As used herein, the term "veterinary chemical" refers to a drug or medicine used to treat or prevent disease, injury and pests in animals. Specifically, "veterinary chemicals" may refer to antiparasitic drugs, antiviral drugs, feed additives, water additives, disinfectants, glutaraldehyde, formalin, mixtures thereof, and the like. In one example, the feed additive may be anticoccidial drugs or ionophores. Water additives refer to chemicals that can be added to the water lines of terrestrial animals instead of to the feed. Veterinary chemicals can be administered to terrestrial animals by any method known in the art. Specifically, veterinary chemicals refer to at least one antimicrobial drug, such as an antiparasitic drug, an antiviral drug or an antibiotic.

[0056] The test animal used in the method according to any aspect of the present invention can be contacted with both the antibiotic and the veterinary chemical simultaneously and / or successively. Changes in the environment within the test animal lead to epigenetic changes, and this can be determined using the method according to any aspect of the present invention.

[0057] Specifically, in the method according to any aspect of the present invention, the methylation status of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 CpG sites is determined in step (a). Those skilled in the art can determine the number of CpG sites to be used in step (a) according to any aspect of the present invention. Even more specifically, according to the method according to any aspect of the present invention, the methylation status of at least two CpG sites is determined in step (a).

[0058] The method according to any aspect of the present invention further comprises the following steps: (i) subjecting the DNA sample to bisulfite modification before step (a).

[0059] "Bisulfite treatment" of genomic DNA used interchangeably with the term "bisulfite modification" refers to processing genomic DNA with a deaminating agent (e.g., bisulfite), which can be used to process all DNA, whether or not it is methylated. Specifically, the term "bisulfite" as used herein encompasses any suitable type of bisulfite, such as sodium bisulfite, or other chemical reagents, which can chemically convert cytosine (C) to uracil (U) without chemically modifying methylated cytosine, and therefore can be used to differentially modify DNA sequences based on the methylation state of DNA, such as U.S. Patent Publication US2010 / 0112595. As used herein, the reagent of "differential modification" methylated or non-methylated DNA encompasses any reagent that modifies methylated and / or non-methylated DNA in the following method, and produces a distinguishable product from methylated and non-methylated DNA by the method, thereby allowing identification of DNA methylation state. Such methods may include, but are not limited to, chemical reactions (e.g., C to U conversion by bisulfite) and enzymatic treatment (e.g., by the cutting of methylation-dependent endonucleases). Thus, an enzyme that preferentially cleaves or digests methylated DNA is one that is able to cleave or digest a DNA molecule with much greater efficiency when the DNA is methylated, whereas an enzyme that preferentially cleaves or digests unmethylated DNA exhibits significantly greater efficiency when the DNA is not methylated.

[0060] In the context of the present invention, any "non-bisulfite-based method" and "non-bisulfite-based quantitative method" are also included to test the methylation status at any given methylation site to be tested. Such terms refer to any method for quantifying methylated or non-methylated nucleic acids that does not require the use of bisulfite. These terms also refer to methods for preparing nucleic acids to be quantified that do not require bisulfite treatment. Examples of non-bisulfite-based methods include, but are not limited to, methods for digesting nucleic acids using one or more methylation-sensitive enzymes and methods for separating nucleic acids using reagents that bind nucleic acids based on methylation status. The terms "methyl-sensitive enzyme" and "methylation-sensitive restriction enzyme" are DNA restriction endonucleases whose activity depends on the methylation status of their DNA recognition sites. For example, there are methyl-sensitive enzymes that cut or digest at their DNA recognition sequences only when their DNA recognition sequences are not methylated. Therefore, unmethylated DNA samples will be cut into smaller fragments than methylated DNA samples. Similarly, highly methylated DNA samples will not be cut. In contrast, there are methyl-sensitive enzymes that cut at their DNA recognition sequences only when their DNA recognition sequences are methylated. As used herein, the terms "cleavage," "shearing," and "digestion" are used interchangeably.

[0061] Therefore, prior to performing step (a) according to any aspect of the present invention, the genomic DNA contained in / obtained or extracted from the cells is first subjected to bisulfite treatment.

[0062] Alternative methods available in the art may be used instead of bisulfite treatment. The skilled person will understand which other methods are used. In one example, TET-assisted pyridine borane sequencing (TAPS) can be used to detect 5mC and 5hmC (Yibin Liu et al., Nature Biotechnology, 37: 424-429 (2019)).

[0063] The cells used according to any aspect of the present invention are obtained from a biological sample selected from blood, brain, sperm, and any other tissue or sample that provides genomic DNA for use in the method according to any aspect of the present invention. Specifically, the biological sample can include any biological material containing DNA obtained from a subject, and can be liquid, solid, or both, can be tissue or bone, or body fluids such as blood, lymph, etc. Specifically, the biological sample that can be used in the present invention can include biological cells or fragments thereof.

[0064] According to a further aspect of the present invention, there is provided a method of determining whether a test animal and / or a test animal from which a product is derived has been treated and / or is currently receiving treatment with at least one antibiotic, and if so, determining the unique class of antibiotic with which the test animal has been treated and / or is currently receiving treatment, the method comprising:

[0065] (a) determining a test methylation profile from genomic material, wherein the genomic material is contained in a biological sample obtained from a test animal and / or an animal-derived product sample; and

[0066] (b) comparing the test methylation profile obtained from (a) to one or more predetermined reference methylation profiles, wherein each predetermined reference methylation profile is from a different animal of the same biological taxonomic group as the test animal, and each of said different animals is treated with a different class of antibiotics,

[0067] wherein if the test methylation profile of (a) is significantly similar to one of the predetermined reference methylation profiles, it is confirmed that the test animal has been treated with the same class of antibiotics as the animal having the predetermined reference profile, and it is confirmed that the test animal has received and / or is currently receiving treatment with the unique class of antibiotics; and wherein the test animal is a terrestrial animal.

[0068] The unique classes of antibiotics are amide alcohols, aminocyclins, aminoglycosides, ansamycins, beta-lactams, carbacephems, carbapenems, cephalosporins, chloramphenicol, fluoroquinolones, glycopeptides, glycylcyclines, ketolides, lincosamides, lipopeptides, macrolides, monocyclic beta-lactams, nitrofurans, nitroimidazoles, oxazolidinones, penicillins, phosphonic acid derivatives, pleuromutilins, polymyxins, polypeptides, quinolones, rifamycins, clofazimines, steroidal antibacterials, streptogramins, sulfonamides, tetracyclines and trimethoprim.

[0069] In one example, a set of predetermined reference spectra may be prepared for different animals for use as controls, wherein each animal has been exposed to a different class of antibiotics and / or each part of the animal (i.e. tissue, muscle, blood, skin) has its own unique predetermined reference methylation profile, which also forms part of the predetermined reference profile set. For each part of an animal or animal-derived product used as genomic material, a different animal may have its own set of predetermined reference profiles, wherein the different animals are from the same biological taxonomic group as the test animal, wherein each was treated with a different class of antibiotics. For example, each group may be specific for a single animal exposed to a first antibiotic and / or veterinary chemical, wherein each reference profile may be unique for the part of the animal from which the genomic material was extracted. Thus, a set of predetermined reference profiles will be compiled, each set being specific for a control animal of the same biological taxonomic group as the test animal, which control animal is being exposed to or has been exposed to a first, second, third, etc. antibiotic and / or veterinary chemical. When a test methylation profile is obtained from an unknown animal-derived product sample, it is compared to a predetermined set of reference profiles for the same animal taxonomic group as the test animal to determine the unique class of antibiotics and / or veterinary chemicals to which the test animal is or has been exposed.

[0070] Yet another aspect of the invention relates to a method of determining whether a test animal and / or a test animal from which a product is derived has been treated with and / or is currently receiving treatment with at least one antibiotic, and if so, whether the antibiotic is used as a growth promoter or a therapeutic agent, the method comprising:

[0071] (a) determining a test methylation profile from genomic material contained in a biological sample obtained from a test animal and / or an animal-derived product; and

[0072] (b) comparing the test methylation profile obtained from (a) to one or more predetermined reference methylation profiles, wherein each predetermined reference methylation profile is from a different control animal of the same biological taxonomic group as the test animal, and each different control animal has been treated with an antibiotic as a growth promoter or therapeutic agent,

[0073] wherein if the test methylation profile of (a) is significantly similar to one of the predetermined reference methylation profiles, it is confirmed that the test animal is being treated with the antibiotic in the same manner as a control animal having a similar predetermined reference profile, and it is confirmed that the test animal has received and / or is currently receiving treatment with the antibiotic as a growth promoter or therapeutic agent; and wherein the test animal is a terrestrial animal.

[0074] As used herein, the term "growth promoter" refers to antibiotics used to help improve animal production efficiency by increasing weight gain and product yield. Antibiotics can be used as growth promoters as opposed to as therapeutic agents (ie, for treating a disease).

[0075] According to a further aspect of the present invention, there is provided a method for determining whether, prior to obtaining a product, a test animal from which the product was obtained had undergone a withdrawal period without treatment with at least one antibiotic and / or veterinary chemical, the method comprising:

[0076] (a) determining a test methylation profile from genomic material contained in a biological sample obtained from a product of animal origin; and

[0077] (b) comparing the test methylation profile obtained from (a) to at least two predetermined reference methylation profiles, wherein at least one predetermined reference methylation profile is from a control animal that has undergone a withdrawal period and another predetermined reference methylation profile is from a control animal of the same biological taxonomic group as the test animal that has not undergone a withdrawal period prior to obtaining the product,

[0078] wherein if the test methylation profile of (a) is significantly similar to a predetermined reference methylation profile, then determining whether the test animal has experienced a drug withdrawal period; and

[0079] The test animal is a terrestrial animal.

[0080] As used herein, the term "withdrawal period" refers to the period from the point in time when the antibiotic and / or veterinary chemical is no longer supplied to the animal to the point in time when the remaining antibiotic is broken down in the body until it becomes a non-functional agent and is eventually excreted from the animal. The withdrawal period for different antibiotics can vary from 1 or 2 days to several weeks. A "withdrawal" period is required from the time the antibiotic is administered to the time the animal is legally slaughtered or the product is obtained from the animal. Therefore, the time required for the body to break down the antibiotic until it is no longer functional or no longer exists is called the withdrawal time (or withdrawal period). Once the withdrawal period has passed, the antibiotic has been eliminated from the animal's system.

[0081] According to another aspect of the present invention, there is provided the use of DNA methylation profiling for determining whether a test animal and / or a test animal from which a product is derived has been treated with and / or is currently receiving treatment with at least one antibiotic and / or veterinary chemical, wherein the test animal is a terrestrial animal and the veterinary chemical is an antiparasitic drug, an antiviral drug, a feed additive, a water additive, a disinfectant, glutaraldehyde and / or formalin.

[0082] In one example, the veterinary chemical is at least one antimicrobial drug, such as an antiparasitic drug, an antiviral drug, or an antibiotic.

[0083] According to any aspect of the present invention, a test methylation profile and a reference methylation profile can be obtained by determining the methylation status of a set of preselected CpG sites in the genome of the test and reference animals, respectively. The CpG sites according to any aspect of the present invention can be selected from the following Table A. Specifically, the CpG sites according to any aspect of the present invention are at least one CpG site selected from the following Table A.

[0084] Table A: CpG sites in test or reference animals that are differentially methylated in animals exposed to antibiotics and / or veterinary chemicals.

[0085]

[0086] Example

[0087] The foregoing describes preferred embodiments, which, as will be appreciated by those skilled in the art, may be subject to changes or modifications in design, construction or operation without departing from the scope of the claims. For example, these changes are intended to be covered by the scope of the claims.

[0088] Example 1: In vitro study of the effects of three different antibiotics on the amount of DNA methylation in a porcine intestinal cell line.

[0089] Immortalized cell lines from porcine intestine (IPEC2-J2ACC 701 from DSMZ cell culture) at passage 7 were used. DPBSD8537 (Sigma-Aldrich) was used in 12-well flat-bottom plates at 1×10 4 Cells / well were seeded and initially incubated for 4 days. Following incubation, cells were treated with one of the following four treatments for up to an additional 4 days:

[0090] 1) Incubate for 4 days without any antibiotics

[0091] 2) Incubate with antibiotics for 1 day, then incubate in antibiotic-free medium for 1-3 days

[0092] 3) Incubate with antibiotics for 2 days, then incubate in antibiotic-free medium for 1-2 days

[0093] 4) Incubate with antibiotics for 3 days, then incubate in antibiotic-free medium for 1 day.

[0094] Treatments 2, 3, and 4 were repeated with 3 different antibiotic treatments; 200 mg / L tetracycline hydrochloride, 100 mg / L amoxicillin, and 480 mg / L colistin. Cells were harvested at each time point for each treatment in duplicate. The media was removed, the cells were washed once with DPBS, and the cells were detached from the bottom of the wells using a cell scraper. The cell pellets were then stored at -80°C until ready for use. DNA was isolated using the DNA Stool kit (Macherey and Nagel). The methylation level of 100 ng of isolated DNA was determined using the Methylated DNA Quantification Kit (Sigma-Aldrich), which compares the sample to a standard curve of known methylated DNA.

[0095] Table 1: Average amount of methylated DNA isolated from IPEC2 cells (ng / 100 ng isolated DNA) incubated for 4 days in medium without antibiotics or in medium containing 200 mg / L tetracycline hydrochloride (Tet), 100 mg / L amoxicillin (Amx), or 480 mg / L colistin (Col).

[0096]

[0097] 1 represents the standard error of the mean reported

[0098] 2 NA, not applicable

[0099] 3 Standard error of the mean could not be calculated because insufficient DNA was isolated from one of the two replicates.

[0100] The results showed that all three antibiotics caused a decrease in global DNA methylation within 24 hours of treatment. When compared to control cells without antibiotics, the effect was most pronounced in cells treated with tetracycline, followed by amoxicillin, and lastly in cells treated with colistin. After washing the cells and replacing the antibiotic-treated medium with antibiotic-free medium, differential methylation was also partially restored within 1-3 days. The results suggest that antibiotic treatment can alter the methylation state of eukaryotic cells, and that these effects may be specific to the antibiotic class and duration of treatment.

[0101] Example 2: Effects of three classes of antibiotics administered as growth promoters or therapeutic agents (with or without a withdrawal period) on the methylome of broiler chickens.

[0102] A study was conducted to determine whether methylation of CpG sites in the genome of chicken breast tissue and blood would be differentially affected by:

[0103] 1) Administer antibiotics to promote growth

[0104] 2) Administration of antibiotics as therapeutic agents

[0105] 3) Types of topical antibiotics

[0106] 4) Administer systemically absorbed antibiotics

[0107] 5) Administration of therapeutic doses of antibiotics, with or without a withdrawal period prior to slaughter.

[0108] To create the treatment regimen for the in vivo experiments in chickens, three different antibiotics were utilized (Table 2).

[0109] Table 2: Antibiotic class, mode of action, absorption level and withdrawal period for the 3 antibiotics used to create the treatment regimen in the in vivo study in chickens.

[0110]

[0111] A chicken experiment was conducted, which was carried out from the day of hatching to the 42nd day to represent the average slaughter age of European broilers. A total of 870 Ross308 male broilers were divided into 58 ground pens, each containing 15 chickens. A broiler feed based on corn-soybean meal was formulated to meet all nutritional requirements of the initial (0-14 days), growth (14-28 days) and fattening (28-42 days) feeding stages. The treatment scheme was created by adding different amounts of antibiotics to the basal feed for different durations (Table 3).

[0112] Table 3: Description of the control group and the groups given antibiotic treatment in the 42-day broiler trial.

[0113]

[0114] On days 28 (treatments 1-3) and 42 (all treatments), two animals per pen were sacrificed for sampling to extract blood and various tissues, including pectoralis major muscle tissue. A total of 96 samples were then collected for analysis using the Invitrogen TM Purelink TM DNA was isolated using the GENE DNase Mini Kit (ThermoFisher Scientific, Waltham, MA, USA) and subsequently subjected to reduced bisulfite sequencing to compare the methylation levels of CpG sites in the genome:

[0115] Day 28, breast and blood from 4 birds / treatment = (4 birds x 3 treatments) x (2 tissues / bird) = 24 samples

[0116] Day 42, breast and blood from 5 birds / treatment = (5 birds x 7 treatments) x (2 tissues / bird) = 70 samples

[0117] Day 42, additional blood samples from treatment regimen 1 and treatment regimen 3 = 2 samples

[0118] Total = 96 samples.

[0119] Chicken blood and breast tissue samples were prepared using the Zymo RRBS protocol. The resulting libraries were sequenced and mapped to the red junglefowl (gallus gallus) 5.0 genome using the tool bsmap 2.9.

[0120] Based on the resulting methylation ratios, all CpGs were retained that had non-zero coverage in all individual samples for a particular tissue. This yielded 1,078,713 CpGs for breast and 1,077,240 CpGs for blood.

[0121] To further analyze the ability to classify samples treated with different antibiotics based on methylation data, the R package randomForest was used and a random forest-based classification was built using the 100,000 most variable CpGs in each tissue.

[0122] First, the CpG set was divided into 10,000 chunks, and each chunk was used to classify the sample into different categories using random forests. This was repeated 100 times. All CpGs with a positive "mean reduction in precision" score in a single classification were summed up, and the top 10,000 CpGs with the highest frequency of occurrence were retained. A second random forest was then built, using these 10,000 selected CpGs as features and classified. This was repeated 100 times, and the average classification error as well as the best classification error were evaluated.

[0123] Results showed no differences in broiler growth (weight gain, feed intake or feed conversion ratio) between the different antibiotic treatment groups. This is likely due to the very clean conditions and optimal nutrition the birds received during the trial, as antibiotics were not required to improve growth performance or prevent disease. Therefore, there was no need to correct for the weight or health of the birds when comparing CpG site methylation between treatments.

[0124] The results of random forest analysis of CpG methylation showed that the tylosin-withdrawal group and the amoxicillin-treated group were distinguishable from the control group, with an error rate of <0.05 when considering the best classification (Table 4). The BMD-treated group and the tylosin-treated group without a withdrawal period were also distinguishable from the control group, although the best classification error rates in these treatment groups were slightly higher, ranging from 0.056 to 0.111.

[0125] Table 4: Random forest analysis of CpG methylation based on the 100,000 most variable CpGs in genomic DNA samples from breast tissue of antibiotic treated broiler chickens compared to controls.

[0126]

[0127]

[0128] When random forest analysis was performed on the 100,000 most variable CpGs in blood samples from different treatment regimens, the best classification error rate for distinguishing each antibiotic treatment group from the control group was below 0.100 (Table 5).

[0129] The positions of cytosine residues that were differentially methylated in antibiotic-treated chickens compared to control chickens are listed for breast (Table 6) and blood (Table 7).

[0130] These results indicate that treatment of broiler chickens with antibiotics leads to significant changes in CpG methylation profiles and can therefore be used to differentiate between antibiotic-free and antibiotic-treated poultry, regardless of antibiotic class, purpose of use, or withdrawal period used.

[0131] Table 5: Random forest analysis of CpG methylation based on the 100,000 most variable CpGs in blood genomic DNA samples from antibiotic treated broiler chickens compared to controls.

[0132]

[0133] Table 6: Genomic sites of differentially methylated CpG cytosine residues in blood-extracted DNA from the control group compared to each of the individual antibiotic treatment groups.

[0134]

[0135] Table 7: Genomic sites of differentially methylated CpG cytosine residues in DNA extracted from blood of the control group compared to each antibiotic treatment group.

[0136]

[0137] Example 3: Effects of three classes of anticoccidial drugs on the methylome of broiler chickens.

[0138] Modern broiler chickens are commonly affected by protozoan parasites of the genus Eimeria, which cause an intestinal disease known as coccidiosis. Therefore, coccidiostats are commonly used in broiler management to prevent and control coccidiosis.

[0139] A study was conducted to determine whether methylation of CpG sites in the genome of chicken breast tissue and blood is differentially affected by administration of different classes of anticoccidial agents and whether the resulting epigenetic signatures could be used to create analytical tools to measure anticoccidial drug use in broiler chickens from meat products.

[0140] A chicken experiment was conducted from hatching day to day 42 to represent the average slaughter age of European broilers. A total of 400 Ross308 male broilers were divided into 40 ground pens, each containing 10 chickens. A broiler feed based on corn-soybean meal was formulated to meet all nutritional requirements for the initial (0-14 days), growth (14-28 days) and fattening (28-42 days) feeding stages. A treatment regimen was created by adding different anticoccidial treatments to broilers (Table 7).

[0141] Table 7: Description of the control and anticoccidial treatment groups in the 42-day broiler experiment.

[0142]

[0143] On days 28 and 42, two animals per pen were sacrificed for sampling to extract blood and various tissues, including pectoralis muscle tissue (pectoralis major). TM Purelink TM A total of 160 samples were used for DNA isolation using the GENE DN A mini kit (Thermo Fisher Scientific, Waltham, MA, USA) and subsequently subjected to reduced bisulfite sequencing to compare the methylation levels of CpG sites in the genome.

[0144] Chicken blood and breast tissue samples were prepared using the Zymo RRBS protocol. The resulting libraries were sequenced and mapped to the Red Junglefowl 5.0 genome using the tool bsmap 2.9. Based on the resulting methylation ratios, all CpGs were retained that had non-zero coverage in all individual samples for a particular tissue.

[0145] To further analyze the ability to classify samples treated with different anticoccidial agents based on methylation data, the R package random forest was used and a random forest-based classification was built using the 100,000 most variable CpGs in each tissue.

[0146] First, the CpG set was divided into 10,000 chunks, and each chunk was used to classify the sample into different categories using random forests. This was repeated 100 times. All CpGs with a positive "mean reduction in precision" score in a single classification were summed up, and the top 10,000 CpGs with the highest frequency of occurrence were retained. A second random forest was then built, using these 10,000 selected CpGs as features and classified. This was repeated 100 times, and the average classification error as well as the best classification error were evaluated.

[0147] The results showed that treatment of broiler chickens with anticoccidial drugs resulted in significant changes in the CpG methylation profile, which can therefore be used to distinguish between meat from poultry that did not contain anticoccidial drugs and meat from poultry that was treated with anticoccidial drugs. The results also suggest that epigenetic signatures in broiler chicken tissues can be used to distinguish the class of anticoccidial drugs used.

Claims

1. A method for determining whether a test animal and / or a test animal from which a product is derived has received and / or is currently receiving treatment with at least one antibiotic and / or veterinary chemical, the method comprising: (a) determining a test methylation profile from genomic material contained in a biological sample obtained from a test animal and / or an animal-derived product; and (b) comparing the test methylation profile obtained from (a) with a reference methylation profile obtained from a control animal of the same biological taxonomic group as the test animal, wherein the control animal has not been and / or is not currently being treated with at least one antibiotic and / or veterinary chemical, wherein a difference in the test methylation profile of (a) compared to the reference methylation profile from the control animal indicates that the test animal has received and / or is currently receiving treatment with at least one antibiotic and / or veterinary chemical; and wherein the test animal is a terrestrial animal, and The veterinary chemicals are antiparasitic drugs, antiviral drugs, feed additives, water additives, disinfectants, glutaraldehyde and / or formalin.

2. The method according to claim 1, wherein the difference is hypomethylation or hypermethylation.

3. A method for determining whether a test animal and / or a test animal from which a product is derived has received and / or is currently receiving treatment with at least one antibiotic and / or veterinary chemical, the method comprising: (a) determining the methylation status of at least one CpG site in genomic material contained in a biological sample obtained from a test animal, (b) comparing the methylation status of the CpG sites from (a) with the methylation status of a control animal of the same taxonomic group as the test animal, wherein the control animal has not been and / or is not currently being treated with at least one antibiotic and / or veterinary chemical, wherein a difference in the test methylation status of (a) in the test animal compared to the CpG site in the control animal indicates that the test animal has received and / or is currently receiving treatment with at least one antibiotic and / or veterinary chemical; and wherein the test animal is a terrestrial animal, and The veterinary chemicals are antiparasitic drugs, antiviral drugs, feed additives, water additives, disinfectants, glutaraldehyde and / or formalin.

4. The method according to claim 3, wherein the difference in methylation status is hypomethylation or hypermethylation of CpG sites in the test animal, and the hypomethylation or hypermethylation of the CpG sites indicates that the test animal has received and / or is currently receiving treatment with at least one antibiotic and / or veterinary chemical.

5. The method according to claim 3 or 4, wherein the CpG site is at least one CpG site selected from the following table: 。 6. The method according to any of the preceding claims, wherein the product of animal origin is meat, processed meat, milk, collagen, feathers, dairy products, blood and / or bone.

7. The method according to any of the preceding claims, wherein the animal-derived product is from a terrestrial animal selected from the group consisting of chicken, lamb, cow, goat, sheep, pig, horse, donkey, turkey, duck, goose, quail, rabbit and mule.

8. A method according to any one of the preceding claims, wherein the biological sample is selected from the group consisting of blood, brain, sperm, milk and any other tissue or sample providing genomic DNA.

9. The method according to any one of the preceding claims, wherein the antibiotic is selected from the class of amide alcohols, aminocyclins, aminoglycosides, ansamycins, beta-lactams, carbacephems, carbapenems, cephalosporins, chloramphenicol, fluoroquinolones, glycopeptides, glycylcyclines, ketolides, lincosamides, lipopeptides, macrolides, monocyclic beta-lactams, nitrofurans, nitroimidazoles, oxazolidinones, penicillins, phosphonic acid derivatives, pleuromutilins, polymyxins, polypeptides, quinolones, rifamycins, clofazimines, steroidal antibacterials, streptogramins, sulfonamides, tetracyclines and trimethoprim.

10. A method according to any one of the preceding claims, wherein the feed additive is an anticoccidial drug or an ionophore.

11. A method of determining whether a test animal and / or a test animal from which a product is derived has received and / or is currently receiving treatment with at least one antibiotic, and if so, determining the unique class of antibiotic with which the test animal has been treated and / or is currently receiving treatment, the method comprising: (a) determining a test methylation profile from genomic material, wherein the genomic material is contained in a biological sample obtained from a test animal and / or an animal-derived product sample; and (b) comparing the test methylation profile obtained from (a) to one or more predetermined reference methylation profiles, wherein each predetermined reference methylation profile is from a different animal of the same biological taxonomic group as the test animal, and each of said different animals is treated with a different class of antibiotics, wherein if the test methylation profile of (a) is significantly similar to one of the predetermined reference methylation profiles, then it is confirmed that the test animal has been treated with the same class of antibiotics as the animal having the predetermined reference profile, and it is confirmed that the test animal has received and / or is currently receiving treatment with antibiotics of the unique class; and The test animal is a terrestrial animal.

12. The method according to claim 10, wherein the unique class of antibiotics is amide alcohols, aminocyclins, aminoglycosides, ansamycins, beta-lactams, carbacephems, carbapenems, cephalosporins, chloramphenicol, fluoroquinolones, glycopeptides, glycylcyclines, ketolides, lincosamides, lipopeptides, macrolides, monocyclic beta-lactams, nitrofurans, nitroimidazoles, oxazolidinones, penicillins, phosphonic acid derivatives, pleuromutilins, polymyxins, polypeptides, quinolones, rifamycins, clofazimines, steroidal antibacterials, streptogramins, sulfonamides, tetracyclines, and trimethoprim.

13. A method of determining whether a test animal and / or a test animal from which a product is derived has received and / or is currently receiving treatment with at least one antibiotic, and if so, whether the antibiotic is used as a growth promoter or a therapeutic agent, the method comprising: (a) determining a test methylation profile from genomic material contained in a biological sample obtained from a test animal and / or an animal-derived product; and (b) comparing the test methylation profile obtained from (a) to one or more predetermined reference methylation profiles, wherein each predetermined reference methylation profile is from a different control animal of the same biological taxonomic group as the test animal, and each different control animal has been treated with an antibiotic as a growth promoter or therapeutic agent, wherein if the test methylation profile of (a) is significantly similar to one of the predetermined reference methylation profiles, it is confirmed that the test animal is being treated with the antibiotic in the same manner as a control animal having a similar predetermined reference profile, and it is confirmed that the test animal has received and / or is currently receiving treatment with said antibiotic as a growth promoter or therapeutic agent; and The test animal is a terrestrial animal.

14. A method for determining whether, prior to obtaining a product, a test animal from which the product was obtained had undergone a withdrawal period without treatment with at least one antibiotic and / or veterinary chemical, the method comprising: (a) determining a test methylation profile from genomic material contained in a biological sample obtained from a product of animal origin; and (b) comparing the test methylation profile obtained from (a) to at least two predetermined reference methylation profiles, wherein at least one predetermined reference methylation profile is from a control animal that has undergone a withdrawal period and another predetermined reference methylation profile is from a control animal of the same biological taxonomic group as the test animal that has not undergone a withdrawal period prior to obtaining the product, wherein if the test methylation profile of (a) is significantly similar to a predetermined reference methylation profile, then determining whether the test animal has experienced a drug withdrawal period; and wherein the test animal is a terrestrial animal, and The veterinary chemicals are antiparasitic drugs, antiviral drugs, feed additives, water additives, disinfectants, glutaraldehyde and / or formalin.

15. Use of DNA methylation profiling to determine whether a test animal and / or a test animal from which a product is derived has received and / or is currently receiving treatment with at least one antibiotic and / or veterinary chemical, wherein the test animal is a terrestrial animal and the veterinary chemical is an antiparasitic drug, an antiviral drug, a feed additive, a water additive, a disinfectant, glutaraldehyde and / or formalin.

Images