Methods for determining resistance or sensitivity of pigs to bacterial infection

Abstract

To provide DNA markers associated with bacterial infections including mycoplasmal pneumonia, porcine pleuropneumonia, and small intestine infection, and to provide applications thereof.SOLUTION: A method for determining resistance or sensitivity of pigs to bacterial infection comprises analyzing any one gene selected from the group consisting of FCGR2 gene, LRRFIP1 gene, STING1 gene, IFI16 gene, CD14 gene, RKDC gene, TRIM40 gene, CGAS gene, RIGI gene, MX1 gene, TRIM21 gene, and MSR1 gene in a genome of a target pig. The method further comprises analyzing the genome of the target pig for at least one single nucleotide polymorphism (SNP) selected from the group consisting of 1 to 17 in the table to determine whether the pig is a resistant type or a sensitive type.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to a method for determining resistance and susceptibility of pigs to bacterial infection. The present invention is useful in animal hygiene management, diagnosis and treatment, animal breeding, pig farming, etc. [Background technology]

[0002] There are many diseases that reduce productivity in the pig farming industry, but bacterial diseases such as mycoplasmal pneumonia, porcine pleuropneumonia, and enteritis are major diseases that affect many pigs and cause secondary infections with other bacteria and viruses, resulting in poor growth and reduced feed efficiency. Although vaccines exist, these diseases have not yet been eradicated, and with the use of antibiotics being restricted worldwide, genetic improvement of pigs' own disease resistance is expected as a new countermeasure.

[0003] A QTL associated with mycoplasmal pneumonia has been detected on chromosome 2, and a resistant population has been created (Non-Patent Document 1). In addition, it has been reported that NLRP3-2906, an existing disease resistance improvement DNA marker, is associated with mycoplasmal pneumonia, and NOD2-2197 and TLR5-1205 are associated with porcine pleuropneumonia (Non-Patent Document 2).

[0004] Meanwhile, two DNA markers on chromosome 13 associated with porcine circovirus type 2 resistance have been identified by genome-wide association study (GWAS) (Patent Document 1). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent Publication No. 2021-171055 [Non-patent literature]

[0006] [Non-Patent Document 1] Okamura T et al (2012) A genome-wide scan for quantitative trait loci affecting respiratory disease and immune capacity in Landrace pigs. Anim. Genet. 43, 721-729. doi: 10.1111 / j.1365-2052.2012.02359. [Non-patent document 2] Suzuki K et al (2022) Polymorphisms in pattern recognition receptor genes are associated with respiratory disease severity in pig farms. Animals 12, 3163. doi:10.3390 / ani12223163 Summary of the Invention [Problem to be solved by the invention]

[0007] The results of QTL analysis do not reveal the responsible genes or mechanisms, and it is difficult to apply the results to other populations, so they cannot be used as general-purpose DNA markers. However, it would be desirable to have DNA markers associated with bacterial infections, including mycoplasmal pneumonia, porcine pleuropneumonia, and small intestine inflammation. [Means for solving the problem]

[0008] The present application provides the following: [1] A method for determining resistance or susceptibility of pigs to bacterial infection, comprising analyzing any of the following genes selected from the FCGR2 gene, LRRFIP1 gene, STING1 gene, IFI16 gene, CD14 gene, PRKDC gene, TRIM40 gene, CGAS gene, RIGI gene, MX1 gene, TRIM21 gene, and MSR1 gene in the genome of the target pig. [2] The method for distinguishing between the above items 1 and 12, wherein the genome of a target pig is analyzed to determine whether it is resistant or susceptible for at least one single nucleotide polymorphism (SNP) selected from the group consisting of 1 to 17 in the table below.

[0009] JPEG2025156087000002.jpg119170[3] A discrimination method according to 1 or 2, in which a target pig is discriminated as being resistant to bacterial infection if at least one SNP in the genome of the target pig is of the resistant type in the table. [4] A method for determining whether a bacterial infection is present in a pig or pigs, comprising the steps of: (a) detecting a bacterial infection in a pig or pigs with a bacterial pathogen selected from the group consisting of mycoplasmal pneumonia, porcine pleuropneumonia, salmonellosis, atrophic rhinitis, swine erysipelas, enteritis including proliferative enteritis, edema disease, and Glaesser's disease; [5] The method of any one of 1 to 4, wherein the bacterial infection is an infection with at least one bacterium selected from Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, Salmonella cholerae suis, Salmonella typhimurium, Bordetella bronchiseptica, Pasteurella multocida, Erysipelothrix rhudiopathiae, Lawsonia intracellularis, Escherichia coli, and Haemophilus parasuis. [6] An oligonucleotide complementary to a portion of any one of the base sequences set forth in SEQ ID NOs: 1 to 17 or their complementary sequences, and having a length of 15 or more bases, for use in the discrimination method described in any one of 1 to 5. [7] A method for breeding pigs resistant to bacterial infection, comprising selecting pigs resistant to bacterial infection using the discrimination method described in any one of 1 to 6. [8] A pig breeding method, comprising selecting pigs resistant to bacterial infection using the discrimination method described in any one of 1 to 7, and using the selected pigs or their offspring for breeding. [9] A method for producing pigs resistant to bacterial infection, comprising selecting pigs resistant to bacterial infection using the discrimination method described in any one of 1 to 8, and using the selected pigs or their offspring for breeding.

[0010] The present invention also provides the following: [1] A method for determining resistance or susceptibility of pigs to bacterial infection, comprising analyzing any gene selected from the group consisting of the FCGR2 gene, LRRFIP1 gene, STING1 gene, IFI16 gene, CD14 gene, PRKDC gene, TRIM40 gene, CGAS gene, RIGI gene, and MX1 gene in the genome of a target pig. [2] The method described in 1, wherein at least one single nucleotide polymorphism (SNP) selected from the group consisting of 1 to 13 in the table below in the genome of the target pig is analyzed to determine whether it is resistant or susceptible.

[0011] JPEG2025156087000003.jpg93170[3] A method according to 1 or 2, wherein the target pig is determined to be resistant to bacterial infection if the genome of the target pig has at least one SNP of the resistance type in the table. [4] The method according to any one of 1 to 3, wherein the bacterial infection is an infection with at least one causative bacterium selected from mycoplasma pneumonia, porcine pleuropneumonia, salmonellosis, atrophic rhinitis, swine erysipelas, proliferative enteritis, edema disease, and Glaesser's disease. [5] The method for determining bacterial infection described in 1, wherein the bacterial infection is an infection with at least one bacterium selected from Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, Salmonella cholerae suis, Salmonella typhimurium, Bordetella bronchiseptica, Pasteurella multocida, Erysipelothrix rhudiopathiae, Lawsonia intracellularis, Escherichia coli, and Haemophilus parasuis. [6] An oligonucleotide for use in the discrimination method described in any one of 1 to 5, which is complementary to a portion of any one of the base sequences set forth in SEQ ID NOs: 1 to 12 or their complementary sequences and has a length of 15 or more bases. [7] A method for breeding pigs resistant to bacterial infection, comprising selecting pigs resistant to bacterial infection using the discrimination method described in any one of 1 to 5. [8] A pig breeding method, comprising selecting pigs resistant to bacterial infection using the discrimination method described in any one of 1 to 5, and using the selected pigs or their offspring for breeding. [9] A method for producing pigs resistant to bacterial infection, comprising selecting pigs resistant to bacterial infection using the discrimination method described in any one of 1 to 5, and using the selected pigs or their offspring for breeding. [Effects of the Invention]

[0012] The DNA markers provided in this application enable the selection of pigs associated with resistance or susceptibility to mycoplasmal pneumonia, porcine pleuropneumonia, and enteritis in three-way crossbred pigs. The ability to select appropriate breeding pigs is expected to reduce pig losses due to pneumonia and enteritis in the pig farming industry and reduce hygiene costs such as vaccines and antibiotics.

[0013] The DNA markers provided by this application were not obtained by analyzing traditional pig breed populations such as Landrace or Duroc. Therefore, the developed DNA markers are not limited to specific pig breeds, and are expected to be effective for three-way crossbred pigs, which are the final product for consumers.

[0014] Because the responsible gene has been identified in this study, it will be useful for functional analysis at the molecular and cellular levels where DNA markers exert their effects. By clarifying the comparative mechanism, it is expected that the applicability and appeal of this method will increase. [Brief explanation of the drawings]

[0015] [Figure 1] The outline of the mycoplasmal pneumonia score and porcine pleuropneumonia score (box plot) for the four pig populations used in the analysis, as well as the number of pigs with / without small intestinal lesions, are shown. [Figure 2]The results of the association analysis between pneumonia scores and the presence or absence of small intestinal inflammation lesions and SNP genotypes in the above four populations are shown in graphs to show the differences in effect between populations for each detected gene / SNP. [Figure 3] The base sequences surrounding the identified SNPs (sequences of SEQ ID NOS: 1 to 17). The positions of the polymorphisms are indicated in brackets [ ]. DETAILED DESCRIPTION OF THE INVENTION

[0016] When a numerical range is expressed as "m to n," the range includes the endpoints m and n, unless otherwise specified.

[0017] [Discrimination method, polymorphic markers] This embodiment provides a method for identifying pigs with high resistance to bacterial infection (sometimes referred to as bacterial infection-resistant pigs) by detecting polymorphisms present in the pig genome. Resistance to bacterial infection includes direct and indirect traits. Direct immune traits include resistance to bacterial infection, low bacterial infection load in the blood, resistance to onset of disease, and resistance to progression to severe disease if disease does occur. Indirect traits refer to traits such as cytokine production ability and cytokine receptor gene production ability that do not directly support bacterial infection resistance in pigs, but from which a relationship between the trait and bacterial infection resistance can be inferred.

[0018] In this embodiment, unless otherwise specified, the term "bacteria" refers to pathogenic bacteria capable of infecting pigs, including at least one causative bacteria of bacterial infection selected from mycoplasma pneumonia, porcine pleuropneumonia, salmonellosis, atrophic rhinitis, swine erysipelas, enteritis including proliferative enteritis, edema disease, and Glaesser's disease.

[0019] More specifically, bacteria in this embodiment include Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, Salmonella cholerae suis, Salmonella typhimurium, Bordetella bronchiseptica, Pasteurella multocida, Erysipelothrix rhudiopathiae, Lawsonia intracellularis, Escherichia coli, and Haemophilus parasuis.

[0020] The determination method of this embodiment can be suitably used to determine whether or not a subject is resistant to infection with bacteria that cause mycoplasmal pneumonia, porcine pleuropneumonia, and small intestine inflammation. In this embodiment, the term "bacterial infection" includes not only a state in which a subject is infected with bacteria and develops infectious symptoms, but also a state in which no infectious symptoms are developed.

[0021] Mycoplasmal pneumonia in pigs is a respiratory disease caused by the bacterium Mycoplasma hyopneumoniae. The incidence of mycoplasmal pneumonia in pigs is extremely high worldwide. Even in the absence of obvious clinical symptoms, mycoplasmal-infected pigs can cause economic losses due to growth retardation and reduced feed efficiency. Mycoplasmal-infected pigs also have a weakened biological defense mechanism, making them more susceptible to infection with other bacteria or viruses. Therefore, mycoplasmal pneumonia in pigs is a very important underlying disease of porcine respiratory complex. Veterinarians and other professionals can determine whether or not a target pig (i.e., a pig used in the discrimination method of this embodiment) has mycoplasmal pneumonia in pigs. If necessary, lungs can be removed from slaughtered pigs and scored for lesions in the right anterior lobe, right middle lobe, left anterior lobe, and left middle lobe to indicate the severity of the disease.

[0022] Porcine pleuropneumonia is a respiratory disease caused by the bacterium Actinobacillus pleuropneumoniae. Infection can result in death, exacerbation of disease due to multiple infections, and poor growth, resulting in significant economic damage to fattening pig production. The causative bacteria are classified into serotypes 1 to 12, with outbreaks occurring in Japan in the order of serotypes 2, 5, and 1. The determination method of this embodiment can be effective against bacteria of any serotype. Veterinarians can determine whether a pig has porcine pleuropneumonia. This determination can be based on the degree of adhesion between the lungs and the diaphragm, etc. Porcine pleuropneumonia is also an inspection item during the meat distribution process, so manuals for veterinarians at slaughterhouses are available. The diagnosis method and procedures are well known to those skilled in the art.

[0023] In the case of small intestine inflammation in pigs, the most suspected cause is infection with Lawsonia intracellularis, a bacterium that causes proliferative enteritis. Other possible causes include Clostridium, Salmonella, and rotavirus. Small intestine inflammation is seen in the middle to late stages of fattening, and various symptoms occur, including bloody stools, anemia, and poor growth, but in severe cases, it can even cause death. It is an inspection item during the process of distribution as meat, and if a veterinarian at a slaughterhouse diagnoses "small intestine inflammation," some or all of the internal organs are discarded.

[0024] In this embodiment, when referring to pigs, the breed is not particularly limited. The discrimination method of this embodiment can be applied to pigs of Duroc, Large Yorkshire, Landrace, Berkshire, Hampshire, and crossbreeds thereof. Crossbreeds include ordinary pigs (three-way crossbreed pigs).

[0025] Discrimination includes determining with a technically significant degree of certainty whether the target pigs are resistant to bacterial infection, and determining with a technically significant degree of certainty whether the target pigs are susceptible. Discrimination is also referred to as identification, appraisal, differentiation, judgment, testing, etc. The certainty of discrimination can be improved by considering the type, number, and combination of polymorphisms used.

[0026] Discrimination can be achieved by detecting whether an individual is resistant or susceptible at a specific polymorphic site. A polymorphism refers to the existence of individuals with different genotypes within a population of the same species, or the different gene or DNA sequences. Polymorphisms include single nucleotide polymorphisms (SNPs), insertion / deletion polymorphisms, restriction fragment length polymorphisms, variable number of tandem repeats (VNTRs), hypervariable regions, minisatellites, dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats, and simple sequence repeats.

[0027] A single nucleotide polymorphism (SNP) occurs when a single nucleotide is replaced with another nucleotide (a nucleotide, or a di-, tetra-, oligo-, or polynucleotide) at a polymorphic site consisting of a single nucleotide. A single nucleotide polymorphism can also occur when a single nucleotide is deleted or inserted compared to a reference allele. When describing a polymorphism, the type of nucleotide involved may be conveniently indicated by the type of base (A, G, T, or C) that constitutes that nucleotide.

[0028] In the discrimination method of this embodiment, SNPs present in the pig genome are used as markers, and the SNPs in this embodiment are related to any of the following genes: FCGR2 gene, LRRFIP1 gene, STING1 gene, IFI16 gene, CD14 gene, PRKDC gene, TRIM40 gene, CGAS gene, RIGI gene, MX1 gene, TRIM21 gene, and MSR1 gene.

[0029] Specifically, the discrimination method of this embodiment discriminates resistance / susceptibility according to the table below for at least one polymorphism selected from the table below.

[0030] JPEG2025156087000004.jpg77170

[0031] In the table, dbSNPID is a unique number in the SNP database managed by the National Center for Biotechnology Information (NCBI).

[0032] The above polymorphisms are expected to have a more potent effect on pneumonia resistance than at least NLRP3-2906 and NOD2-2197, among NLRP3-2906, NOD2-2197, and TLR5-1205 described in Non-Patent Document 2. In the current analysis of the association between SNPs and pneumonia severity, the present inventors simultaneously input NLRP3-2906, NOD2-2197, and TLR5-1205 into a model in addition to the above-mentioned novel marker group, and detected a significant difference in TLR5-1205, but not in NLRP3-2906 or NOD2-2197.

[0033] Of the above polymorphisms, polymorphisms in the FCGR2 gene and LRRFIP1 gene have been found to be associated with both mycoplasmal pneumonia and porcine pleuropneumonia in multiple analyzed populations (Figure 2). Therefore, the impact of these polymorphisms is significant, and it is expected that they will have a uniform effect across various populations with different sanitary environments.

[0034] Whether the above polymorphisms are resistant to the listed polymorphisms can be determined by various polymorphism analysis methods well known to those skilled in the art, including direct sequencing, SNP Type Assay (in which a common tag sequence is added to the primer during PCR amplification, and a universal fluorescent primer that anneals to this sequence is used for detection), TaqMan® PCR (SNP typing using a real-time PCR system), RFLP, PCR-SSCP, ASO hybridization, ARMS, denaturing gradient gel electrophoresis, RNase A cleavage, chemical cleavage, DOL, Invader, MALDI-TOF / MS, TDI, molecular beacon, dynamic allele-specific hybridization, padlock probe, UCAN, nucleic acid hybridization using a DNA chip or DNA microarray, and ECA.

[0035] Samples used for polymorphism analysis include, for example, hair roots, blood, meat pieces, and semen from the subject pig. DNA can be extracted from these samples by an appropriate method and used for polymorphism analysis.

[0036] [Primers, probes, kits] This embodiment relates to an oligonucleotide, a composition containing the oligonucleotide (e.g., a solution containing such an oligonucleotide and a buffer, or a frozen product thereof), and a kit for use in identifying pigs resistant to the above-mentioned bacterial infection.

[0037] The oligonucleotides may be PCR primers, primer pairs, probes, or sets thereof for detecting the above polymorphisms.

[0038] When the oligonucleotide is a PCR primer for detecting a polymorphism, its length is usually 15 to 100 bases, preferably 17 to 30 bases. When the oligonucleotide is a probe for detecting a product amplified by PCR, its length is usually 15 to 100 bases, preferably 17 to 30 bases. Primers and probes can be designed appropriately with reference to conventional primer and probe design methods. Commercially available software for primer design methods can also be used in this embodiment. Oligonucleotides used as primers or probes can be prepared, for example, using a commercially available oligonucleotide synthesizer.

[0039] The probe is preferably labeled. As a labeling method, the 5' end of the polynucleotide is labeled with T4 polynucleotide kinase. 32 Labeling by phosphorylation with P, or by using a DNA polymerase such as Klenow enzyme and a random hexamer polynucleotide as a primer. 32 Examples of such methods include a method of incorporating a substrate base labeled with an isotope such as P, a fluorescent dye, or biotin (random prime method, etc.).

[0040] The composition containing an oligonucleotide of this embodiment may contain, in addition to the above-mentioned oligonucleotide, sterilized water, a pH buffer, a stabilizer, and the like.

[0041] The kit of this embodiment contains, in addition to the above-mentioned oligonucleotides and compositions, for example, dNTPs (deoxynucleotide triphosphates), a thermostable DNA polymerase, Mg 2+ , PCR buffer, control standards, reaction vessels, and instructions. The kit may also include equipment for specimen collection.

[0042] In the above-mentioned identification or discrimination reagent (test drug of this embodiment), in addition to the polynucleotide, which is the active ingredient, for example, sterilized water, saline, vegetable oil, surfactant, lipid, solubilizing agent, buffer, protein stabilizer (such as BSA or gelatin), preservative, etc. may be mixed as needed.

[0043] [Breeding method] As described above, this embodiment provides a method for distinguishing pigs that are resistant to bacterial infection. Therefore, this embodiment makes it possible to predict pigs that have a target trait (resistance to bacterial infection) at an early stage, which is useful for efficiently creating a pig population or line that has a desired trait. Therefore, this embodiment provides a method for breeding pigs that are resistant to bacterial infection, which includes selecting pigs that are resistant to bacterial infection using the discrimination method of this embodiment.

[0044] As a pig breeding method for selecting and raising or breeding pigs with a predetermined trait, for example, by mating a bacterial infection-resistant pig selected by this embodiment or its progeny with a pig having a certain desirable trait (e.g., high meat production capacity, robustness, high reproductive capacity, etc.), a pig line having the certain trait and being resistant to bacterial infection can be created. Thus, this embodiment provides a breeding method for bacterial infection-resistant pigs, which includes selecting bacterial infection-resistant pigs by the discrimination method of this embodiment and using the selected pigs or their progeny for mating, and a production method for bacterial infection-resistant pigs, which includes selecting bacterial infection-resistant pigs by the discrimination method of this embodiment and using the selected pigs or their progeny for mating. In addition, breeding pigs produced by the method for producing bacterial infection-resistant pigs, which includes selecting pigs that are resistant to bacterial infection using the discrimination method of this embodiment and using the selected pigs for breeding, to obtain progeny, is also included in the scope of the production method of this embodiment, as long as progeny that are resistant to bacterial infection associated with the polymorphic markers provided by this embodiment are obtained, regardless of whether or not bacterial infection-resistant pigs are selected for the progeny using the discrimination method of this embodiment.

[0045] [Verification of polymorphism effects, etc.] Whether the polymorphisms disclosed herein have the desired effect can be verified by various methods. For example, verification can be performed by isolating cells with different genotypes from blood and observing their response to bacterial infection. Verification can also be performed by investigating the association between polymorphisms and immune function or disease-related traits (e.g., pneumonia incidence rate) using a pig population resistant to bacterial infection and a population other than the base population. Such verification methods are well known to those skilled in the art, and those skilled in the art can design and carry out experiments as appropriate.

[0046] This embodiment provides a pig herd with a high proportion of individuals with a genotype that is resistant to bacterial infection. In this embodiment, unless otherwise specified, the term "herd" in relation to pigs refers to a group of at least 10 pigs that are kept so as to be distinguishable from other individuals or groups.

[0047] The present embodiment also relates to a method for producing a bacterial infection-resistant pig by the method of the present embodiment, using breeding materials (e.g., selected semen, We provide frozen semen, as well as meat and meat products (e.g., ham, sausage, bacon, roast pork, etc.). [Example]

[0048] [Analysis 1: Genotyping in the analyzed population] A total of 862 pigs (three-way crossbred pigs) were analyzed from general farms in Gifu Prefecture, including Farm A (280 pigs) and Farm B (279 pigs) sampled in 2016, and Farm A (178 pigs) and Farm C (125 pigs) sampled in 2019. Data on mycoplasmal pneumonia scores, porcine pleuropneumonia scores, and the presence or absence of small intestinal lesions were collected at the slaughterhouse. Data summaries are shown using boxplots for pneumonia and by number of pigs for small intestinal lesions (Figure 1).

[0049] Mycoplasma pneumonia score: Also known as the Goodwin score. The liver lesions in the lung lobes are scored on a scale of 0 to 55, with 10 points for the right anterior lobe, right middle lobe, left anterior lobe, and left middle lobe, and 5 points for the right posterior lobe, accessory lobe, and left posterior lobe. Pig pleuropneumonia score: The degree of adhesion between the lungs and the thoracic cavity, diaphragm, etc. is evaluated on a scale of 0 to 4. Presence or absence of small intestinal inflammation lesions: If thickening of part or all of the small intestine is found at the slaughter inspection facility, it is evaluated as "lesion present," and if not, it is evaluated as "no lesion."

[0050] From the pig genome database Sscrofa11.1 (https: / / asia.ensembl.org / Sus_scrofa / Info / Index?db=core;g=ENSSSCG00000005503;r=1:258044610-258058970), information on 62 single nucleotide polymorphisms (SNPs) with amino acid substitutions in 23 immune-related genes that may be associated with susceptibility / resistance to pneumonia and enteritis was extracted, and genotypes were determined in the four populations mentioned above using the SNP Type Assay method (Standard Biotools) (Table 1).

[0051] [Table 1] JPEG2025156087000006.jpg197170JPEG2025156087000007.jpg234170

[0052] For some of the analyzed populations, information on SNPs associated with amino acid substitutions in other immune-related genes was extracted from the pig genome database Sscrofa11.1, and genotypes were determined by sequencing.

[0053] [Analysis 2: Association analysis between amino acid polymorphisms and pneumonia scores] In the four populations mentioned above, the association between pneumonia score and the presence or absence of small intestinal lesions and SNP genotypes was analyzed by univariate analysis (analysis of variance for mycoplasmal pneumonia, Kruskal-Wallis test for porcine pleuropneumonia, and chi-square test for small intestinal lesions). The calculated significance probability (P) and values ​​where P < 0.2 were found are indicated by an asterisk (*) (Table 2).

[0054] [Table 2]

[0055] For SNPs with P < 0.2, the allele frequencies in each population are shown in Table 3 (mycoplasmal pneumonia score), Table 4 (pig pleuropneumonia score), and Table 5 (presence or absence of small intestinal inflammation lesions). In addition, when there were multiple SNPs with P < 0.2 within the same gene (shown as squares in Table 2), they were expected to be inherited in a linked manner, so haplotype estimation was performed, and the allele frequencies are shown in Tables 3, 4, and 5.

[0056] [Table 3] JPEG2025156087000010.jpg230170

[0057] [Table 4]

[0058] [Table 5] JPEG2025156087000013.jpg118170

[0059] For SNPs and haplotypes with a P < 0.2, the association between pneumonia score and genotype was analyzed using a generalized linear mixed model, and the association between the presence or absence of small intestinal lesions and genotype was analyzed using a stepwise logistic regression analysis. The effects of genotype on mycoplasmal pneumonia (Table 6), porcine pleuropneumonia (Table 7), and small intestinal inflammation (Table 8) were estimated, taking into account the effects of sex and sampling date. P < 0.05 is indicated by *, and P < 0.01 is indicated by **. The differences in the effects between populations for each detected gene / SNP are also shown graphically (Figure 2).

[0060] [Table 6] JPEG2025156087000015.jpg173170

[0061] [Table 7]

[0062] [Table 8]

[0063] Excluding overlaps, we identified 17 SNPs in 12 genes as amino acid polymorphisms associated with pneumonia scores and the presence or absence of small intestinal lesions (candidate DNA markers for improved disease resistance), and summarized them by distinguishing between resistant and susceptible alleles (Table 9). These genes have the function of recognizing common molecules that make up bacteria and activating the immune response, and may affect resistance / susceptibility not only to mycoplasmal pneumonia, porcine pleuropneumonia, and small intestinal inflammation, but also to other bacterial infections.

[0064] [Table 9]

[0065] dbSNPID is an ID in the SNP database managed by the National Center for Biotechnology Information (NCBI).

[0066] The nucleotide sequences surrounding the identified SNPs were obtained from Sscrofa11.1 and are shown in the sequence listing as SEQ ID NOs: 1 to 17.

[0067] [Sequence listed in the sequence listing] SEQ ID NO:1 Surrounding sequence of rs320243268 SEQ ID NO:2 Surrounding sequence of rs331355666 SEQ ID NO:3 Surrounding sequence of rs319566914 SEQ ID NO:4 Surrounding sequence of rs81218215 SEQ ID NO:5 Surrounding sequence of rs80915627 SEQ ID NO:6 Surrounding sequence of rs81218902 SEQ ID NO:7 Surrounding sequence of rs80900450 SEQ ID NO:8 rs327309867 surrounding sequence SEQ ID NO:9 Surrounding sequence of rs339299789 SEQ ID NO:10 Surrounding sequence of rs329876422 SEQ ID NO:11 Surrounding sequence of rs334436029 SEQ ID NO:12 Surrounding sequence of rs81214124 SEQ ID NO:13 Surrounding sequence of rs55618275 SEQ ID NO:14 Surrounding sequence of rs81411505 SEQ ID NO:15 Surrounding sequence of rs340330924 SEQ ID NO:16 Surrounding sequence of rs80855305 SEQ ID NO:17 rs325112499 surrounding sequence

Claims

1. A method for determining resistance or susceptibility of pigs to bacterial infection, comprising analyzing any of the following genes selected from the FCGR2 gene, LRRFIP1 gene, STING1 gene, IFI16 gene, CD14 gene, PRKDC gene, TRIM40 gene, CGAS gene, RIGI gene, MX1 gene, TRIM21 gene, and MSR1 gene in the genome of the target pig.

2. The method according to claim 1, wherein at least one single nucleotide polymorphism (SNP) selected from the table below in the genome of a target pig is analyzed to determine whether the pig is resistant or susceptible.

3. A discrimination method as described in claim 2, in which the target pig is discriminated as being resistant to bacterial infection if at least one SNP in the genome of the target pig is of the resistance type in the table.

4. The method of claim 1, wherein the bacterial infection is an infection with at least one causative bacterium selected from the group consisting of mycoplasmal pneumonia, porcine pleuropneumonia, salmonellosis, atrophic rhinitis, swine erysipelas, small intestine including proliferative enteritis, edema disease, and Glaesser's disease.

5. The method of claim 1, wherein the bacterial infection is an infection with at least one bacterium selected from Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, Salmonella cholerae suis, Salmonella typhimurium, Bordetella bronchiseptica, Pasteurella multocida, Erysipelothrix rhudiopathiae, Lawsonia intracellularis, Escherichia coli, and Haemophilus parasuis.

6. An oligonucleotide having a length of 15 or more bases, which is complementary to a part of any one of the base sequences set forth in SEQ ID NOs: 1 to 17 or its complementary sequence, for use in the discrimination method according to any one of claims 1 to 5.

7. A method for breeding pigs resistant to bacterial infection, comprising selecting pigs resistant to bacterial infection by the discrimination method according to any one of claims 1 to 5.

8. A pig breeding method, comprising selecting pigs resistant to bacterial infection by the discrimination method according to any one of claims 1 to 5, and using the selected pigs or their progeny for breeding.

9. A method for producing pigs resistant to bacterial infection, comprising selecting pigs resistant to bacterial infection by the discrimination method according to any one of claims 1 to 5, and using the selected pigs or their progeny for breeding.

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