Detection of the use of antibiotics and / or chemicals in aquatic animals using epigenetic means

Abstract

The present invention is a method 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 being treated with at least one antibiotic and / or veterinary chemical substance, comprising: (a) determining a test methylation profile from genomic material contained in a biological sample obtained from the test animal and / or an animal-derived product; (b) comparing the test methylation profile obtained from step (a) with a reference methylation profile obtained from a control animal of the same biological taxon as the test animal, wherein the control animal has not been treated with and / or is not currently being treated with at least one antibiotic and / or veterinary chemical substance; and a difference in comparing the test methylation profile of step (a) with the reference methylation profile from the control animal indicates that the test animal has been treated with and / or is currently being treated with at least one antibiotic and / or veterinary chemical substance; relating to a method, wherein the test animal is an aquatic animal and the veterinary chemical substance is an antiparasitic agent, antiviral agent, feed additive, water additive, disinfectant, glutaraldehyde and / or formalin used in aquaculture.

Description

Technical Field

[0001] The present invention relates to a method for detecting the use and / or administration of at least one antibiotic and / or chemical substance to aquatic animals using epigenetic means. In particular, the method determines the methylation state of CpG sites in test animals and compares the resulting methylation state with a reference methylation state of control animals reared without the use or administration of antibiotics and / or chemical substances, thereby detecting the use of at least one antibiotic or the administration of at least one antibiotic and / or chemical substance during the rearing of at least one aquatic animal.

Background Art

[0002] Antibiotics are used in the treatment of bacterial diseases in human and veterinary medicine. Antibiotics have also been used prophylactically at low levels in feed or water to improve the growth rate and mortality of livestock. However, the excessive use of antibiotics, particularly in agriculture, is associated with an increase in antibiotic-resistant bacteria and ultimately impairs the ability to treat bacterial diseases in humans and animals alike. Furthermore, the use of antibiotics has been associated with other unintended and undesirable physiological outcomes such as low birth weight and increased disease susceptibility in infants from mothers treated with antibiotics. Therefore, tools for assessing not only current but also past antibiotic use will be of high interest in livestock production for the purpose of guiding breeding, feeding, veterinary and / or management practices. In addition, the analysis of meat and meat products for assessing past antibiotic use in individual animals prior to slaughter may be of high interest for the auditing, certification and labeling of meat products.

[0003] To date, antibiotic testing has involved chemical assays to find residues of antibiotics present in an animal's blood or tissue. However, most antibiotics used for growth promotion are added to feed or water, and most of these classes are poorly absorbed in the gastrointestinal system, so residue testing in blood or tissue does not capture the use of all antibiotic classes. There is currently no way to test aquatic animals or meat products for previous antibiotic use unless antibiotic residues remain in the residue and do not remain in the animal (i.e., blood or tissue) or the aqueous environment (i.e., pond or tank).

[0004] Accordingly, there is a need in the art for simple and accurate means of detecting current and past antibiotic and / or drug use in animals, particularly aquatic farm animals.

[0005] Epigenetics is the study of inherited traits caused by mechanisms other than changes in the underlying DNA sequence. In other words, epigenetic marks "direct" our genes. Epigenetic marks can be either chemical (e.g., methylation), protein-based (e.g., 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 by the next generation as a form of epigenetic memory. Since DNA methylation events have been shown to be regulatory mechanisms associated with gene silencing, expression, chromatin remodeling, or imprinting, these changes may be the cause of genetic changes in gene activity. Epigenetics is attractive for animal husbandry because it can identify the causal relationships and heritability of complex traits and diseases. DNA methylation patterns are modified throughout an individual's life by environmental forces such as diet, stress, drugs, or pollution. Some environments are more likely to increase specific methylation patterns, and these patterns can contribute to epigenetic and / or phenotypic variation between individuals.

[0006] Several studies have shown that antibiotic exposure can alter epigenetic markers in eukaryotic cells. For example, in a study examining the relationship between antibiotic use during pregnancy and birth weight outcomes, specific regions of the genome that were differentially methylated in response to antibiotic exposure were found (Vidal et al., 2013). In studies conducted using cultured plant cells, changes in global DNA methylation were observed when the cells were exposed to the antibiotic kanamycin (Bardini et al., 2003). However, epigenetic changes that may occur due to current and / or past use of antibiotics and / or drugs in animals, particularly aquatic animals, have not been mentioned in the art.

[0007] Therefore, there is still a need in the art for a simple and accurate method for detecting the current and / or past use of antibiotics and / or drugs in animals, particularly aquatic animals.

BRIEF DESCRIPTION OF THE INVENTION

[0008] The present invention solves the above problems by providing a means for developing a novel analytics for evaluating the use of antibiotics or other chemicals in aquatic agricultural animal production, meat, and / or meat products using alterations in DNA methylation patterns. In particular, this means compares the methylation status of at least one CpG site in a test animal from which a test animal and / or product is derived with the methylation status of the corresponding CpG site in a control animal from which a control animal and / or product is derived, to detect the use of antibiotics and / or veterinary chemicals in a test animal from which a test animal and / or product is derived, wherein no antibiotics and / or veterinary chemicals are used in the control animal, and the presence of hypomethylation or hypermethylation at the CpG site of the test animal indicates that the test animal has been treated with antibiotics and / or veterinary chemicals, including the method. The method according to any aspect of the present invention is a simple and accurate method for detecting the current and / or past use of antibiotics and / or drugs in animals, particularly aquatic animals.

[0009] The present invention is based on the finding that for animals, contact with antibiotics and / or veterinary chemicals can cause stress to the internal environment of the animal, which can permanently change the animal's genome epigenetically. In particular, 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. In particular, the present invention is based on the finding that contact with antibiotics and / or veterinary chemicals can also cause changes in the epigenetic mechanisms of animals, 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 found that by utilizing this property, it is possible to identify a genomic "epigenetic fingerprint" specific to the presence of antibiotics and / or veterinary chemicals in not only one animal but also, as far as possible, all animals that have been brought into contact with at least one of the antibiotics and / or veterinary chemicals. For example, these "epigenetic fingerprints" can be specific not only to animals that have been brought into contact with antibiotics and / or veterinary chemicals but also to any product derived from that animal (individual). Based on these findings, the present invention provides a means for detecting the use of antibiotics and / or veterinary chemicals in the cultivation of animals, particularly aquatic animals, such as the aquatic animals from which animal-derived products are derived. In this way, by using the method according to any aspect of the present invention, it is possible to accurately and reliably determine whether any test animal or its product has been brought into contact with or treated with at least one antibiotic and / or veterinary chemical. Subsequently, the method according to any aspect of the present invention can also distinguish the prophylactic use of antibiotics (growth promotion) from the therapeutic use of antibiotics using DNA methylation analysis. DNA methylation analysis can also be a step in elucidating whether an animal treated with a veterinary chemical and / or an antibiotic has been given a withdrawal period from the veterinary chemical and / or the antibiotic before the animal is killed or recovered for use or consumption or to distinguish the use of different classes of antibiotics. The method according to the present invention can also be used to evaluate the current and past use of antibiotics in aquatic livestock animals or meat to investigate epigenetic markers, which can then be used in the preparation of an assay for testing the use of antibiotics in aquatic livestock species. Furthermore, by using the method according to any aspect of the present invention, it is possible to predict and avoid undesirable traits caused by the use of antibiotics and / or veterinary chemicals for animal welfare and more sustainable aquatic livestock production. It is also possible to distinguish the route of administration of antibiotics to animals by using the method according to any aspect of the present invention.

[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 treated with and / or is currently being treated with at least one antibiotic and / or veterinary chemical, comprising: (a) determining a test methylation profile from genomic material contained in a biological sample obtained from the test animal and / or an animal-derived product; (b) comparing the test methylation profile obtained in step (a) with a reference methylation profile obtained from a control animal that has not been treated with and / or is not currently being treated with at least one antibiotic and / or veterinary chemical; wherein a difference in comparing the test methylation profile of step (a) with the reference methylation profile from the control animal indicates that the test animal has been treated with and / or is currently being treated with at least one antibiotic and / or veterinary chemical; the test animal is an aquatic animal, and the veterinary chemical is an antiparasitic agent, antiviral agent, feed additive, water additive, disinfectant, glutaraldehyde and / or formalin used in aquaculture; a method is provided.

[0012] In particular, the method according to any aspect of the present invention can be used to detect differences in antibiotic-related methylation for any stage / age of aquatic animals, particularly aquatic livestock. More specifically, the method according to any aspect of the present invention can track the use of antibiotics and / or veterinary chemicals commonly used in the aquaculture of aquatic animals, particularly aquatic livestock. 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 been treated with and / or is currently being treated with at least one antibiotic and / or veterinary chemical, comprising: (a) determining the methylation status of at least one CpG site in genomic material contained in a biological sample obtained from the test animal; (b) Comparing the methylation status of the CpG sites from step (a) with the methylation status of control animals that have not been treated with and / or are not currently being treated with at least one antibiotic and / or veterinary chemical comprising wherein a difference in comparing the test methylation status of step (a) of the test animal with the CpG sites of the control animals indicates that the test animal has been treated with and / or is currently being treated with at least one antibiotic and / or veterinary chemical wherein the test animal is an aquatic animal and the veterinary chemical is an antiparasitic agent, antiviral agent, feed additive, water additive, disinfectant, glutaraldehyde and / or formalin used in aquaculture a method is provided.

[0013] The difference in methylation status according to any aspect of the present invention is hypomethylation or hypermethylation of the CpG sites of the test animal, and the hypomethylation or hypermethylation of the CpG sites indicates that the test animal has been treated with and / or is currently being treated with at least one antibiotic and / or veterinary chemical.

[0014] As used herein, the term "aquatic animal" refers to any organism that lives entirely or primarily in water, particularly as compared to terrestrial animals. In particular, an aquatic animal according to any aspect of the present invention can be any animal in the animal kingdom that lives primarily in water. These aquatic animals may live in different water forms such as seas, oceans, rivers, lakes, ponds, etc. More specifically, an aquatic animal according to any aspect of the present invention may be any aquatic livestock, or any fish, aquatic mollusk or aquatic crustacean at all life stages including eggs, sperm and gametes. Even more specifically, "aquatic animal" means animals of the following species: (i) fish belonging to the superclass Agnatha and the classes Chondrichthyes, Sarcopterygii and Actinopterygii, (ii) aquatic mollusks belonging to the phylum Mollusca, and (iii) aquatic crustaceans belonging to the subphylum Crustacea. Even more specifically, an aquatic animal according to any aspect of the present invention can be an aquatic animal used in aquaculture. Some non-limiting examples of aquatic animals according to any aspect of the present invention include barramundi, carp, catfish, halibut, marbled crayfish, marine and brackish fish, marine shrimp, mitten crab, mussel, oyster, pangasius, rainbow trout, salmonid, scallop, sea bass, sea bream, soft-shelled crab, soft-shelled turtle, tiger prawn, tilapia, turbot, white-leg prawn, and other decapod crustaceans, bivalves and gastropods.

[0015] As used herein, the term "animal-derived product" refers to products derived from animals, particularly aquatic animals. In particular, the term "test animal-derived product" refers to a sample or subject of a problem to be subjected to the method according to any aspect of the present invention. These products from animals include meat and meat products, and may further include eggs, fats, meat, blood, processed meat and less well-known products, as well as non-food products such as fibers (such as shells, scales, etc.). Animal-derived products may also include products that can be made using livestock products, such as tablets, powders, etc. (e.g., fish oil). In one example, an animal-derived product is meat, eggs, blood, brain, shell, scale, skin, tissue, abdominal muscle tissue, or any other tissue or sample that provides genomic DNA. In particular, an animal-derived product is meat, skin, blood, trimming, or any organ derived from an aquatic animal. In particular, the trimming is used as a by-product for fishmeal / fish oil that is ultimately used in the animal feed industry or for pets. In one example, an animal-derived product sample can be a single type of meat, different types of meat, a single part of a single type of meat, different parts of a single type of meat, or different parts of different types of meat. The sample can be derived from any biological entity having genomic DNA and DNA genomic methylation. In particular, the methylation site is a CpG site.

[0016] As used herein, the term "test" when used in conjunction with the terms "subject" and / or "animal" refers to an entity that is subjected to a method according to any aspect of the invention and that is the basis for the analytical use of the invention. Thus, a "(single) test subject", "(single) test subject group" or "test profile" or "test animal-derived product" is a (single) subject or group of subjects being tested according to the invention, or a profile obtained or generated in this context. In contrast, the terms "reference" or "control" are taken to mainly denote a given entity that is used for comparison with the test entity. For example, the term "reference animal", used interchangeably with "control animal", refers to an animal of the same biological taxonomic group that is used as a comparison or control with respect to a "test animal". Similarly, the terms "sample" and / or "test animal-derived product sample" as used according to any aspect of the invention refer to an entity that can be subjected to the method of the invention. In particular, a sample can be any (test) animal-derived product that can be subjected to the method of the invention to determine whether the test animal from which the test animal-derived product is obtained had or was in contact with at least one antibiotic and / or veterinary chemical. For example, a sample of an animal-derived product can be a fish fillet that is tested according to any aspect of the invention to determine whether the test animal from which the test animal-derived product is obtained was in contact with at least one antibiotic and / or veterinary chemical. In another example, a (test) animal-derived product sample can be a fish egg that is tested according to any aspect of the invention to determine whether the test animal from which the test animal-derived product is obtained is currently in contact with at least one antibiotic and / or veterinary chemical. Blockchain can also be used to make information readily available to consumers.

[0017] As used herein, the term "comprising" should be interpreted to include both "including" and "consisting of", both meanings being specifically intended and thus being individual disclosed aspects of the present invention. As used herein, "and / or" should be interpreted as a specific disclosure of each of two specified features or components, whether or not the other is present. For example, "A and / or B" should be interpreted as (i) A, (ii) B, and (iii) each of A and B, as if each were individually described herein. In the context of the present invention, the terms "about" and "approximately" indicate the range of accuracy understood by a person skilled in the art to still ensure the technical effect of the feature in question. This term typically indicates a deviation of ±20%, ±15%, ±10%, and for example ±5% from the indicated numerical value. As will be understood by a person skilled in the art, the specific deviation of the numerical value of a given technical effect depends on the nature of the technical effect. For example, natural or biological technical effects may generally have such a greater deviation than artificial or engineering technical effects. When an indefinite or definite article, such as "a", "an" or "the", is used to refer to a singular noun, this includes the plural of that noun, unless something else is specifically stated.

[0018] In the context of the present invention, the terms "methylation profile", "methylation pattern", "methylation state", or "methylation status" are used herein to describe the state, situation, or condition of methylation of a genomic sequence, and these terms refer to the characteristics of a DNA segment at a particular genomic locus related to methylation. Such characteristics include, but are not limited to, the presence or absence of methylation of any of the cytosine (C) residues within this DNA sequence, the position of the methylated C residues, the proportion of methylated C's in any particular stretch of residues, and differences in methylation on alleles due to, for example, differences in the origin of the alleles. The DNA segment may also include specific pre-selected methylation sites.

[0019] As used herein, the term "pre-selected methylation site" refers to a methylation site selected from a gene or region that exhibits the highest degree of methylation variation during the training of the method and meets certain quality criteria, such as having a minimum sequencing coverage of 5-fold or more for 5 or more qualified CpG sites. Further, genes having an average methylation level <0.1 or an average methylation level >0.9 can be excluded due to their limited dynamic range. A "reference methylation profile" can be defined based on a plurality of training samples using a multivariate statistical method such as principal component analysis or multidimensional scaling.

[0020] The term "methylation state" refers to the state (i.e., methylation vs. non-methylation) of a particular methylation site, meaning whether the residue or methylation site is methylated or not methylated. Subsequently, a methylation profile can be determined based on the methylation state of one or more methylation sites. Thus, the term "methylation profile" or even "methylation pattern" refers to the relative or absolute concentration of methylated C residues or non-methylated C residues in any particular stretch of residues in the genomic material of a biological sample. For example, if a cytosine (C) residue that is typically not methylated within a DNA sequence is methylated, this may be referred to as "hypermethylation", while if a cytosine (C) residue that is typically methylated within a DNA sequence is not methylated, this may be referred to as "hypomethylation". Similarly, if a cytosine (C) residue within a DNA sequence (e.g., the DNA of a sample nucleic acid from a test subject) is methylated compared to another sequence in a different region or a different individual (e.g., compared to a normal nucleic acid or a standard nucleic acid of a reference sequence), that sequence is considered to be hypermethylated compared to the other sequence. Alternatively, if a cytosine (C) residue within a DNA sequence is not methylated compared to another sequence in a different region or a different individual, that sequence is considered to be hypomethylated compared to the other sequence. These sequences are said to be "differentially methylated". The measurement of the level of differential methylation can be performed by various methods known to those skilled in the art. One method, by way of non-limiting example, is to measure the methylation level of individual aligned CpG sites determined by bisulfite sequencing.

[0021] The term "hypermethylation" refers to an average methylation pattern corresponding to an increase in the presence of 5-mCyt in one or more CpG dinucleotides within the DNA sequence of a test DNA sample compared to the amount of 5-mCyt found in the corresponding CpG dinucleotides in a normal control DNA sample. In particular, the control refers to products derived from aquatic animals or aquatic animals that have not been in contact with antibiotics and / or veterinary chemicals.

[0022] The term "hypomethylation" refers to an average methylation pattern corresponding to a decrease in the presence of 5-mCyt in one or more CpG dinucleotides within the DNA sequence of a test DNA sample, as compared to the amount of 5-mCyt found in the corresponding CpG dinucleotides within a normal control DNA sample. In particular, the control refers to an aquatic animal-derived product or an aquatic animal that has not been in contact with antibiotics and / or veterinary chemicals.

[0023] As used herein, "methylated nucleotide" or "methylated nucleotide base" refers to the presence of a methyl moiety on a nucleotide base, which is not normally present on the recognized typical nucleotide bases. For example, cytosine in its normal form does not contain a methyl moiety in its pyrimidine ring, while 5-methylcytosine contains a methyl moiety at the 5-position of its pyrimidine ring. Thus, cytosine may not be considered a methylated nucleotide in its normal form, and 5-methylcytosine may be considered a methylated nucleotide. In another example, thymine may contain a methyl moiety at the 5-position of its pyrimidine ring, but for the purposes herein, thymine may not be considered a methylated nucleotide when present in DNA. The typical nucleotide bases of DNA are thymine, adenine, cytosine, and guanine. The typical bases of RNA are uracil, adenine, cytosine, and guanine. Correspondingly, a "methylation site" is a position within a target gene nucleic acid region where methylation can occur. For example, a position containing CpG is a methylation site where cytosine may or may not be methylated. In particular, the term "methylated nucleotide" refers to a nucleotide carrying a methyl group attached to the position of the nucleotide available for methylation. These methylated nucleotides are usually found in nature and, to date, methylated cytosines occurring mainly in relation to the dinucleotide CpG but also in relation to CpNpG and CpNpN sequences are considered the most common. In principle, other naturally occurring nucleotides can also be methylated, but these are not considered in any aspect of the present invention.

[0024] As used herein, a "CpG site" or "methylated site" is a nucleotide within a nucleic acid (DNA or RNA) that is susceptible to methylation by either a natural occurring event in vivo or an event initiated to chemically methylate nucleotides in vitro. Some of these sites may be hypermethylated in animals exposed to an antibiotic and / or a veterinary chemical as compared to cells not exposed to the antibiotic and / or the veterinary chemical, and some may be hypomethylated.

[0025] As used herein, a "CpG island" describes a segment of a DNA sequence that contains a CpG density that deviates functionally or structurally. For example, Yamada et al. have described a set of criteria for identifying CpG islands. A CpG island must be at least 400 nucleotides in length, have a GC content of greater than 50%, and an OCF / ECF ratio of greater than 0.6 (Yamada et al., 2004, Genome Research, 14, pp. 247-266). Others have defined less stringent CpG islands as sequences at least 200 nucleotides in length that have a GC content of greater than 50% and an OCF / ECF ratio of greater than 0.6 (Takai et al., 2002, Proc. Natl. Acad. Sci. USA, 99, pp. 3740-3745). In the context of the present invention, the terms "methylation profile", "methylation pattern", "methylation state", or "methylation status" are used herein to describe the state, situation, or condition of methylation of a genomic sequence, and these terms refer to the characteristics of a DNA segment at a particular genomic locus related to methylation. Such characteristics include, but are not limited to, the presence or absence of methylation of any of the cytosine (C) residues within this DNA sequence, the position of the methylated C residues, the proportion of methylated C's in any particular stretch of residues, and differences in methylation on alleles due to, for example, differences in the origin of the alleles.

[0026] As used herein, the term "methylated nucleic acid molecule" refers to a nucleic acid molecule that contains one or more nucleotides that are methylated.

[0027] As used herein, the term "epigenetic change" refers to a chemical (e.g., methylation) change or a protein (e.g., histone) change that occurs to the gene body or its promoter. Due to epigenetic changes, environmental factors such as diet, stress, and prenatal nutrition may affect genes passed from one generation to the next.

[0028] As used herein, the term "genomic material" refers to a nucleic acid molecule or fragment of the genome of a subject or group of subjects. In particular, 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.

[0029] As used herein, a "DNA sample" refers to DNA extracted from a terrestrial animal according to any aspect of the present invention using methods known in the art.

[0030] In particular, when differential methylation is detected in a test animal, i.e., the test animal and / or a product derived from the test animal exhibits hypermethylation or hypomethylation at at least one CpG site as compared to a control (i.e., an aquatic animal that has not been contacted with at least one antibiotic and / or veterinary chemical), the test animal has been or has been in contact with at least one antibiotic and / or veterinary chemical. The difference in methylation according to any aspect of the present invention can be hypomethylation or hypermethylation.

[0031] A biological sample, in the context of the present invention, may include any biological material obtained from a subject or group of subjects containing genomic material, and may be liquid, solid, or both, may be tissue or bone, or may be a body fluid such as blood. In particular, a biological sample useful in the present invention may include living cells or fragments thereof. More specifically, the biological sample is selected from the group consisting of blood, brain, muscle, shell, and any other tissue or sample that provides genomic DNA.

[0032] One or more of the preselected methylation sites in step (a) are methylation sites associated with tissue-specific gene expression, and preferably, the preselected methylation sites are associated with gene expression in one distinct tissue.

[0033] The tissue may be selected from the following.

[0034] (i) A metabolic tissue such as intestinal tissue or abdominal tissue, wherein the intestinal tissue is preferably ileum or jejunum, (ii) Muscle tissue, (iii) Skin tissue, and (iv) Organ tissue.

[0035] As used herein, the term "antibiotic" refers to any medicament that can be administered to terrestrial animals for therapeutic and / or prophylactic purposes. The antibiotic can be administered by any method known in the art. The antibiotic can be orally administered to aquatic animals such that it is ingested in animal feed or in the water in which the aquatic animals are reared, or used as a bath for external infections, according to any aspect of the present invention. In another example, the antibiotic may be injected into the aquatic animals. One of ordinary skill in the art will understand the best way to provide the antibiotic to the animals based on the specific biological taxon of the animal, the type of antibiotic, and the disease to be treated or prevented. In particular, the antibiotic according to any aspect of the present invention can be selected from the group consisting of amphenicol, aminocyclitol, aminoglycoside, ansamycin, beta-lactam, carbaephem, carbapenem, cephalosporin, chloramphenicol, fluoroquinolone, glycopeptide, glycylcycline, ketolide, lincosamide, lipopeptide, macrolide, monobactam, nitrofurans, nitroimidazole, oxazolidinone, penicillin, phosphonic acid derivatives, proionmutilin, polymyxin, polypeptide, quinolone, rifamycin, liminophenazine, steroid antibacterial agents, streptogramin, sulfonamide, tetracycline, and trimethoprim. More specifically, the antibiotic can be selected from the group consisting of tetracycline and fluoroquinolone, particularly norfloxacin.

[0036] In the test animals according to any aspect of the present invention, at least one or more of the above antibiotics may be administered simultaneously or sequentially. Contact of the antibiotics with the aquatic animals can result in epigenetic changes, at least DNA methylation changes, which can be determined using the methods according to any aspect of the present invention. The concentration of the antibiotic at each dose and / or the period during which the antibiotic is administered to the test animal can affect the expansion of differential methylation in the test animal compared to the control animals. Determining the concentration of each dose required by the test animal and the period of antibiotic exposure depending on whether the antibiotic is administered for preventive or therapeutic measures is within the knowledge of those skilled in the art.

[0037] As used herein, the term "veterinary chemical" refers to drugs or medicaments used to treat or prevent diseases, injuries and epizootics in animals in aquaculture. In particular, "veterinary chemicals" can refer to anti-parasitic agents, anti-viral agents, feed additives, water additives, disinfectants, glutaraldehyde, formalin, mixtures thereof, and the like. Veterinary chemicals can be administered to aquatic animals used in aquaculture by any method known in the art.

[0038] The test animals used in the method according to any aspect of the present invention can be contacted with both antibiotics and veterinary chemicals simultaneously and / or sequentially. Changes in the internal environment of the test animals result in epigenetic changes, which can be determined using the methods according to any aspect of the present invention.

[0039] In particular, in the method according to any aspect of the present invention, in step (a), 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. A person skilled in the art will be able to determine the number of CpG sites that need to be used in step (a) according to any aspect of the present invention. Even more specifically, the methylation status of at least two CpG sites is determined in step (a) of the method according to any aspect of the present invention.

[0040] The method according to any aspect of the present invention (i) a step of performing bisulfite modification on a DNA sample before step (a) further comprises.

[0041] "Bisulfite treatment" of genomic DNA, which is used interchangeably with the term "bisulfite modification", refers to the treatment of genomic DNA with a deaminating agent such as bisulfite that can be used to treat all DNA regardless of methylation status. In particular, the term "bisulfite" as used herein refers to any suitable type of bisulfite, such as sodium bisulfite, or other chemical agents that can chemically convert cytosine (C) to uracil (U) without chemically modifying methylated cytosine, and thus can be used to differentially modify DNA sequences based on the methylation state of the DNA. See, for example, U.S. Patent Application Publication No. 2010 / 0112595. As used herein, a reagent that "differentially modifies" methylated DNA or unmethylated DNA includes any reagent that modifies methylated DNA and / or unmethylated DNA in a process where distinguishable products result from methylated DNA and unmethylated DNA, thereby enabling the identification of the DNA methylation state. Such processes can include, but are not limited to, chemical reactions (such as the conversion of C to U by bisulfite) and enzymatic treatments (such as cleavage by methylation-dependent endonucleases). Thus, an enzyme that preferentially cleaves or digests methylated DNA is an enzyme that can cleave or digest DNA molecules with much higher efficiency when the DNA is methylated, while an enzyme that preferentially cleaves or digests unmethylated DNA exhibits significantly higher efficiency when the DNA is not methylated.

[0042] In the context of the present invention, also included are any "non-bisulfite-based methods" and "non-bisulfite-based quantitative methods" for testing the methylation status at any given methylation site being tested. Such terms refer to any method for quantifying methylated or unmethylated nucleic acids that does not require the use of bisulfite. The term also refers to methods for preparing quantified nucleic acids 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 agents that bind to nucleic acids based on their methylation status. The terms "methyl-sensitive enzyme" and "methylation-sensitive restriction enzyme" refer to DNA restriction endonucleases whose activity depends on the methylation pattern of the DNA recognition site. For example, there are methylation-sensitive enzymes that cleave or digest at the DNA recognition sequence only when not methylated. Thus, unmethylated DNA samples are cut into smaller fragments than methylated DNA samples. Similarly, highly methylated DNA samples are not cleaved. In contrast, there are methylation-sensitive enzymes that cleave at the DNA recognition sequence only when methylated. As used herein, the terms "cleave", "cut", and "digest" are used interchangeably.

[0043] Thus, before step (a) according to any aspect of the present invention is carried out, the genomic DNA contained in the cell / obtained or extracted from the cell is first bisulfite-treated.

[0044] Instead of bisulfite treatment, alternative methods available in the art may be used. One skilled in the art will understand which of the other methods to use. In one example, TET-assisted pyridine borane sequencing (TAPS) may be used for the detection of 5mC and 5hmC (Yibin Liu et al., Nature Biotechnology, 37:424-429 (2019)).

[0045] 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 been treated with and / or is currently being treated with at least one antibiotic, and if so, determining a separate class of antibiotics with which the test animal has been treated and / or is currently being treated, the method comprising: (a) determining a test methylation profile from genomic material contained in a biological sample obtained from the test animal and / or an animal-derived product sample; (b) comparing the test methylation profile obtained from step (a) with one or more predetermined reference methylation profiles, each of the predetermined reference methylation profiles being derived from a different animal of the same biological taxon as the test animal, each of the different animals having been treated with a different class of antibiotics; comprising: if the test methylation profile of step (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 been treated with and / or is currently being treated with a separate class of antibiotics; There is provided a method wherein the test animal is an aquatic animal.

[0046] Separate classes of antibiotics are amphenicol, aminocyclitol, aminoglycoside, ansamycin, beta-lactam, carbaephem, carbapenem, cephalosporin, chloramphenicol, fluoroquinolone, glycopeptide, glycylcycline, ketolide, lincosamide, lipopeptide, macrolide, monobactam, nitrofurans, nitroimidazole, oxazolidinone, penicillin, phosphonic acid derivatives, proionmutilin, polymyxin, polypeptide, quinolone, rifamycin, riminophenazine, steroidal antibacterial agents, streptogramin, sulfonamide, tetracycline and trimethoprim.

[0047] In one example, a panel of predetermined reference profiles may be created for different animals used as controls, each animal having been exposed to different classes of antibiotics and / or each part of the animal (i.e., tissue, muscle, blood, skin) having its own unique predetermined methylation reference profile, which also forms part of a panel of predetermined reference profiles. Different animals from the same biological taxon as the test animals, each treated with a different class of antibiotic, may have their own panel of predetermined reference profiles for each part of the animal or animal-derived product used as genomic material. For example, each panel may be specific to a single animal exposed to a first antibiotic and / or veterinary chemical, and each reference profile may be distinct for a part of the animal from which the genomic material is extracted. Thus, a collection of panels of predetermined reference profiles can be created, each panel being specific to one control animal of the same biological taxon as the test animal, the control animal having been or being exposed to first, second, third, etc. antibiotics and / or veterinary chemicals. When a test methylation profile is obtained from an unknown animal-derived product sample, this is compared to different panels of predetermined reference profiles for the same animal taxon as the test animal to determine the distinct class of antibiotics and / or veterinary chemicals to which the test animal has been or is being exposed.

[0048] According to yet another aspect of the present invention, a method of determining whether a test animal and / or a product derived from the test animal has been treated with at least one antibiotic and / or is currently being treated, and if so, determining whether the antibiotic has been used as a growth promoter or as a therapeutic agent, comprising: (a) determining a test methylation profile from genomic material contained in a biological sample obtained from the test animal and / or the animal-derived product; (b) Comparing the test methylation profile obtained from step (a) with one or more predetermined reference methylation profiles, each of the predetermined reference methylation profiles being derived from a different control animal of the same biological taxon as the test animal, each of the different control animals being treated with an antibiotic as a growth promoter or therapeutic agent, and comprising If the test methylation profile of step (a) is significantly similar to one of the predetermined reference methylation profiles, it is confirmed that the test animal has been treated with an antibiotic in the same manner as the control animal having the similar predetermined reference profile was treated, and it is confirmed that the test animal has been treated with an antibiotic as a growth promoter or therapeutic agent and / or is currently being treated; A method is provided wherein the test animal is an aquatic animal.

[0049] As used herein, the term "growth promoter" refers to an antibiotic used to help increase the efficiency of animal production by increasing weight gain and product yield. Antibiotics can be used as growth promoters as opposed to being used as therapeutic agents (i.e., for the treatment of diseases).

[0050] According to a further aspect of the invention, a method for determining whether a test animal from which a product is derived has undergone a withdrawal period without treatment with at least one antibiotic and / or veterinary chemical prior to obtaining the product, (a) Determining a test methylation profile from genomic material contained in a biological sample obtained from an animal-derived product; and (b) Comparing the test methylation profile obtained from step (a) with at least two predetermined reference methylation profiles, at least one of the predetermined reference methylation profiles being derived from a control animal that has undergone a withdrawal period, and the other of the predetermined reference methylation profiles being derived from a control animal of the same biological taxon as the test animal that has not undergone a withdrawal period prior to obtaining the product. comprising If the test methylation profile of step (a) is significantly similar to a predetermined reference methylation profile, it is confirmed that the test animal has or has not undergone a withdrawal period. A method is provided where the test animal is an aquatic animal.

[0051] As used herein, the term "withdrawal period" refers to the period from the time when an animal is no longer given antibiotics and / or veterinary chemicals until the residual antibiotics are broken down in the body into non-functional agents and finally removed from the animal's body. The withdrawal periods for different antibiotics can vary from 1 or 2 days to several weeks. A "withdrawal" period is required before it is legal to slaughter or kill the animal or obtain a product from the animal after the administration of antibiotics. Thus, the time it takes for the body to break down the antibiotics until they are no longer functional or present is called the withdrawal time (or withdrawal period). After the withdrawal period has elapsed, the antibiotics have been removed from the animal's system.

[0052] According to another aspect of the invention, there is provided the use of DNA methylation profiling to determine whether a test animal and / or a test animal from which a product is derived has been treated with and / or is currently being treated with at least one antibiotic and / or veterinary chemical, wherein the test animal is an aquatic animal and the veterinary chemical is an antiparasitic agent, antiviral agent, feed additive, water additive, disinfectant, glutaraldehyde and / or formalin used in aquaculture.

Brief Description of the Drawings

[0053]

Figure 1

Figure 2a

Figure 2b

Figure 3

Figure 4a

Figure 4b

[0054] The above describes preferred embodiments, and these embodiments may be subject to changes or modifications in design, configuration or operation without departing from the scope of the claims, as will be understood by those skilled in the art. These variations are intended to be covered, for example, by the scope of the claims. [Example 1] Mysterious crayfish collected from a population in Lake Reilinger, Germany, were collected for experiments and reared from juveniles in the laboratory. A total of 30 adult mysterious crayfish were reared in one of five treatments using the replicates outlined in Table 1.

[0055] 1) Control - no antibiotic treatment 2) Acute tetracycline administration for 7 days at 10 mg / kg feed 3) Preventive tetracycline administration for 42 days at 5 mg / kg feed 4) Administration for 7 days at 10 mg / kg feed 5) Administration for 28 days at 5 mg / kg feed All other rearing variables remained consistent for all treatment groups. Mysterious crayfish were reared in plastic containers containing tap water at 20 °C, the tap water was changed every two weeks, exposed to a natural day-night light rhythm, and fed daily.

[0056] A subset of abdominal tissue genomic DNA samples (Table 1) was utilized for analysis by the Agilent Sure Select Methyl-Seq Assay. Abdominal tissue was extracted from the mysterious crayfish immediately after the treatment period, and then DNA was extracted as described above (Tonges et al., 2021).

[0057]

Table 1

[0058] Quality control was performed, and the sample concentration was measured with a 2,200 TapeStation (Agilent). The multiplexed samples were submitted to the core facility of DKFZ High Throughput Sequencing and sequenced on a HiSeq system (Illumina). For data analysis, the 361 sequenced genes were mapped to the bisulfite sequencing dataset of the entire marble crab genome (Gatzmann et al., 2018; Xi and Li 2009).

[0059] To identify the antibiotic-specific methylation rate in genomic DNA from abdominal tissue, the average methylation of each sample within 1 kb of the DNA segment was calculated. Next, one-way ANOVA was used to investigate whether the average methylation rate differed significantly between the two treatment groups with tetracycline compared to the control. Analysis comparing the tetracycline treatment to the control yielded 1,754 DNA segments that were significantly differentially methylated (p < 0.05) (Figure 1). PCA shows robust separation between treated and untreated samples and between samples of two different groups treated with tetracycline. Principal component 1 (35.6%) separates the two treatment groups, Tetra A and Tetra B. Furthermore, principal component 2 (32.2%) clearly separates the samples treated with tetracycline from the control (Figure 1).

[0060] The dataset of 1,754 DNA segments was further analyzed, using only CpG positions included in all samples with at least 10 reads. Furthermore, CpGs that were fully methylated or not methylated (average methylation less than 0.2 or greater than 0.8) were excluded. Subsequently, 432 DNA segments with significant differences (p < 0.05) between control and treated animals were revealed by the Kruskal Wallis test.

[0061] Within the five CpGs of the first gene (Scaffold_3671:4528 - 4593), the control samples appear to have a higher overall methylation rate, while the Tetra B group shows the lowest average methylation rate. The nine CpGs of the second gene (Scaffold_690:3807095 - 3807233) have, on average, lower methylation in the control samples. Methylation within the Tetra A group appears increased compared, and the highest methylation rate is seen in the Tetra B group. There is no directional methylation; instead, the differences in methylation are strongly localized (Figure 2b).

[0062] Using the same approach, the antibiotic-specific methylation patterns of different norfloxacin samples were analyzed. The analysis identified 937 different DNA segments that were significantly differentially methylated (p<0.05) (Figure 3). PCA showed robust separation between two different norfloxacin groups as well as between the control and norfloxacin-treated samples. Principal component 1 (42.7%) separates the two treatment groups, NorA and NorB, from each other. Further prominent separation was caused by principal component 2 (30.9%), which shows the difference between the control and the samples treated with norfloxacin (Figure 3).

[0063] The DNA segments of PCA were further analyzed, and only CpG positions with at least 10 reads in all samples were reused. Furthermore, CpGs that were not fully methylated or were methylated (average methylation less than 0.2 or greater than 0.8) were excluded. As a result, 71 DNA segments with significant differences (p<0.05) between control animals and treated animals were revealed by the Kruskal Wallis test.

[0064] Average methylation shows individual DNA segments with moderate methylation differences between the control and the two treatment groups. The effect size was determined using Cohen's d, and four DNA segments showed a small effect (d>0.2), while all others showed a very small effect (d<0.02). Analysis of DMRs revealed no significant differences (p>0.05) between the treatment groups and the control. Nevertheless, treatment-specific differences in DMRs have been demonstrated. These methylation differences are exemplified for the CpGs of two genes (Scaffold_36323:7555-7623, Scaffold_52370:13110767-13110901) (Figure 4).

[0065] In the first gene (Scaffold_36323:7555-7623), CpGs in the control and Nor A groups appear to have, on average, similar methylation rates. On the other hand, when the control group and NorA group were compared with the NorB group, the latter showed higher methylation rates at all CpGs. These differences are particularly evident at CpGs 1-4 and 7 (Figure 4a). The average methylation rate of the second gene (Scaffold_52370:13110767-13110901) did not show a significant difference between the control and NorA groups. On the other hand, the NorB group also showed higher methylation rates at all CpGs in this gene (Figure 4b). The largest difference in methylation can be seen when comparing the control with samples from 4-week treatment with 5 mg / kg norfloxacin (NorB). In both genes, the average methylation increased with treatment. On the other hand, no significant difference from the control was observable with 7-day treatment with 10 mg / kg norfloxacin (NorA). Overall, highly localized methylation patterns can be observed (Figure 4).

[0066] In summary, DNA methylation analysis was able to separate both tetracycline-treated mystery cichlids and norfloxacin-treated mystery cichlids from the control.

Claims

1. 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 being treated with at least one antibiotic and / or veterinary chemical, (a) A step of determining a test methylation profile from genomic material contained in a biological sample obtained from the test animal and / or the animal-derived product, (b) A step of comparing the test methylation profile obtained from step (a) with a reference methylation profile obtained from a control animal of the same biological taxonomy as the test animal, wherein the control animal has not been treated with and / or is not currently treated with at least one antibiotic and / or veterinary chemical, and Includes, The difference between the test methylation profile in step (a) and the reference methylation profile from the control animal indicates that the test animal has been and / or is currently being treated with at least one antibiotic and / or veterinary chemical. The aforementioned test animal is an aquatic animal, A method wherein the veterinary chemical substance is an antiparasitic agent, antiviral agent, feed additive, water additive, disinfectant, glutaraldehyde and / or formalin used in aquaculture.

2. The method according to claim 1, wherein the difference is low methylation or high methylation.

3. 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 being treated with at least one antibiotic and / or veterinary chemical, (a) A step of determining the methylation status of at least one CpG site in the genomic material contained in the biological sample obtained from the test animal, (b) A step of comparing the methylation status of the CpG site in step (a) with the methylation status of a control animal of the same biological taxonomy as the test animal, which has not been treated with and / or is not currently treated with at least one antibiotic and / or veterinary chemical; Includes, The difference between the test methylation status of the test animal in step (a) and the CpG site of the control animal indicates that the test animal has been and / or is currently being treated with at least one antibiotic and / or veterinary chemical. The aforementioned test animal is an aquatic animal, A method wherein the veterinary chemical substance is an antiparasitic agent, antiviral agent, feed additive, water additive, disinfectant, glutaraldehyde and / or formalin used in aquaculture.

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

5. The method according to claim 1 or 2, wherein the animal-derived product is meat, skin, blood, trimmings, or at least one organ from the aquatic animal.

6. The method according to claim 1 or 2, wherein the aquatic animal is selected from the group consisting of fish, crustaceans, and mollusks.

7. The method according to claim 1 or 2, wherein the aquatic animal is selected from the group consisting of carp, salmon, tilapia, catfish, marine fish, brackish water fish, softshell turtle, barramundi, marine shrimp, mitten crab, mystery crayfish, and other decapod crustaceans, bivalves and gastropods.

8. The method according to claim 1 or 2, wherein the biological sample is selected from the group consisting of skin, scales, tissue, abdominal muscle tissue, or any sample that provides genomic DNA.

9. The method according to claim 1 or 2, wherein the antibiotic is selected from the group consisting of amphenicol, aminocyclitol, aminoglycoside, ansamycin, beta-lactam, carbaefem, carbapenem, cephalosporin, chloramphenicol, fluoroquinolone, glycopeptide, glycylcycline, ketride, lincosamide, lipopeptide, macrolide, monobactam, nitrofuran, nitroimidazole, oxazolidinone, penicillin, phosphonic acid derivative, proiromucilin, polymyxin, polypeptide, quinolone, rifamycin, liminofenadine, steroid antibacterial agents, streptogramin, sulfonamide, tetracycline, and trimethoprim.

10. 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 being treated with at least one antibiotic, and if so, for determining the distinct class of antibiotic that the test animal was and / or is currently being treated with, (a) A step of determining a test methylation profile from genomic material contained in a biological sample obtained from the test animal and / or the animal-derived product sample, (b) A step of comparing the test methylation profile obtained from step (a) with one or more predetermined reference methylation profiles, wherein each of the predetermined reference methylation profiles is derived from a different animal of the same biological taxonomy as the test animal, and each of the different animals is treated with a different class of antibiotic, and Includes, If the test methylation profile in step (a) is significantly similar to one of the predetermined reference methylation profiles, it is confirmed that the test animal was treated with the same class of antibiotic to which the animal having the predetermined reference profile was treated, and that the test animal has been treated and / or is currently being treated with the different class of antibiotic, A method wherein the test animal is an aquatic animal.

11. The method according to claim 10, wherein the aforementioned distinct classes of antibiotics are amphenicol, aminocyclitol, aminoglycosides, ansamycin, beta-lactam, carbaefem, carbapenem, cephalosporin, chloramphenicol, fluoroquinolone, glycopeptide, glycylcycline, ketride, lincosamide, lipopeptide, macrolide, monobactam, nitrofuran, nitroimidazole, oxazolidinone, penicillin, phosphonic acid derivatives, proiromucilin, polymyxin, polypeptide, quinolone, rifamycin, liminofenadine, steroid antibacterial agents, streptogramin, sulfonamide, tetracycline, and trimethoprim.

12. A method for determining whether test animals and / or test animals from which a product is derived have been and / or are currently being treated with at least one antibiotic, and if so, whether the antibiotic was used as a growth promoter or therapeutic agent, (a) A step of determining a test methylation profile from genomic material contained in a biological sample obtained from the test animal and / or the animal-derived product, (b) A step of comparing the test methylation profile obtained from step (a) with one or more predetermined reference methylation profiles, wherein each of the predetermined reference methylation profiles is derived from a different control animal of the same biological taxonomy as the test animal, and each of the different control animals is treated with the antibiotic as a growth promoter or therapeutic agent. Includes, If the test methylation profile in step (a) is significantly similar to one of the predetermined reference methylation profiles, the test animal is confirmed to have been treated with the antibiotic in the same manner as the control animal having the similar predetermined reference profile was treated, and the test animal is confirmed to have been treated with and / or currently treated with the antibiotic as a growth promoter or therapeutic agent. The method involves using an aquatic animal as the test animal.

13. A method for determining whether, prior to obtaining the product, the test animals from which the product is derived have undergone a withdrawal period without treatment with at least one antibiotic and / or veterinary chemical, (a) A step of determining a test methylation profile from genomic material contained in a biological sample obtained from the animal-derived product, (b) A step of comparing the test methylation profile obtained from step (a) with at least two predetermined reference methylation profiles, wherein at least one of the predetermined reference methylation profiles is derived from a control animal that has undergone a withdrawal period, and the other of the predetermined reference methylation profiles is derived from a control animal of the same biological taxonomy as the test animal that has not undergone a withdrawal period before the product is obtained. Includes, If the test methylation profile in step (a) is significantly similar to the predetermined reference methylation profile, it is confirmed that the test animal has undergone or has not undergone a withdrawal period. The aforementioned test animal is an aquatic animal, A method wherein the veterinary chemical substance is an antiparasitic agent, antiviral agent, feed additive, water additive, disinfectant, glutaraldehyde and / or formalin used in aquaculture.

14. Use of DNA methylation profiling to determine whether test animals and / or test animals from which a product is derived have been and / or are currently being treated with at least one antibiotic and / or veterinary chemical, wherein the test animals are aquatic animals and the veterinary chemical is an antiparasitic agent, antiviral agent, feed additive, water additive, disinfectant, glutaraldehyde and / or formalin used in aquaculture.

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