Mutant strain of escherichia coli or staphylococcus aureus derived from bovine mastitis and vaccine composition containing same

Abstract

The present invention relates to a mutant strain of Escherichia coli or Staphylococcus aureus derived from bovine mastitis and a vaccine composition containing same. The vaccine composition according to the present invention may be usefully employed to reduce economic losses caused by bovine mastitis and to improve productivity and sustainability across the dairy industry.

Description

E. coli or Staphylococcus aureus mutant strains causing bovine mastitis and vaccine compositions containing the same

[0001] The present invention relates to a vaccine for preventing bovine mastitis, and more particularly, to a mutant strain of Escherichia coli or Staphylococcus aureus caused by bovine mastitis and a vaccine composition containing the same.

[0002] Bovine mastitis is an infectious disease caused by bacterial or fungal invasion of the mammary glands (mammary tissue). It is the most common disease in the dairy industry and causes significant economic losses, including reduced milk production, poor quality, early culling, and increased treatment costs. This has led to a persistent need for the development of effective preventive vaccines or treatments.

[0003] A survey on the isolation frequency of mastitis-causing bacteria in dairy cattle in Korea identified Staphylococcus spp. (24.5%), Escherichia coli (12.9%), Staphylococcus aureus (9.2%), and Streptococcus spp. (7.5%) as the main pathogens. In particular, Staphylococcus aureus was frequently isolated among single species, and E. coli showed the highest isolation rate among Gram-negative bacteria. Therefore, the development of Staphylococcus aureus and E. coli-based vaccines or treatments suitable for the domestic dairy environment is urgent.

[0004] Clinically significant is the marked decline in antibiotic susceptibility to these major pathogens. Staphylococcus aureus showed high resistance to penicillin and ampicillin, while Escherichia coli also showed high resistance to tetracycline, streptomycin, and sulfisoxazole. The rise in these resistant strains is reducing the effectiveness of existing antibiotic treatments, making vaccine strategies to prevent infections more important.

[0005] However, currently commercialized mastitis vaccines have limitations in terms of safety and immune regulation, such as limited efficacy against specific pathogens, excessive inflammatory responses, and low immune persistence. In particular, Staphylococcus aureus exhibits a high degree of immune evasion due to its intracellular parasitism and biofilm formation, while Escherichia coli induces acute inflammatory responses, leading to tissue damage. Therefore, the development of attenuated strain-based vaccines that induce immune stimulation while minimizing pathogenicity and virulence factors is urgently needed.

[0006] Accordingly, the inventors of the present invention developed a vaccine composition using an attenuated mutant strain of E. coli and a mutant strain of Staphylococcus aureus isolated from a cow's mastitis, and completed the present invention by simultaneously securing high safety and effective immune stimulation.

[0007] The purpose of the present invention is to provide a mutant strain of Escherichia coli or Staphylococcus aureus caused by bovine mastitis.

[0008] Another object of the present invention is to provide a vaccine composition for preventing mastitis in cows using the mutant strain.

[0009] To achieve the above purpose,

[0010] The present invention provides an E. coli mutant strain in which an antibiotic resistance gene is inserted into the relA and spoT genes to inactivate their function, and a Staphylococcus aureus mutant strain in which the relQ gene is deleted.

[0011] In addition, the present invention provides a vaccine composition for preventing bovine mastitis comprising the mutant strain.

[0012] The present invention relates to a mutant strain of Escherichia coli or Staphylococcus aureus caused by bovine mastitis and a vaccine composition comprising the same. The vaccine composition according to the present invention can be usefully used to reduce economic losses caused by bovine mastitis and to improve productivity and sustainability of the dairy industry as a whole.

[0013] Figure 1 is a schematic diagram of the construction of an attenuated vaccine seed of EC53 through genetic mutation.

[0014] Figure 2 is a graph showing the results of evaluating the growth ability (growth curve) of an E. colirelAspoT defective mutant.

[0015] Figure 3 is a graph showing the results of evaluating the cell adhesion ability of an E. colirelAspoT defective mutant.

[0016] Figure 4 is a schematic diagram of the production of a Staphylococcus aureus relQ gene deletion mutant using homologous recombination.

[0017] Figure 5 is a graph showing the results of evaluating the growth rate of an outdoor plant and a relQ gene deletion mutant.

[0018] Figure 6 shows the results of evaluating the susceptibility of outdoor strains and relQ gene deletion mutant strains to host-derived antimicrobial peptides (*P < 0.05, **P < 0.01, ***P < 0.001).

[0019] Figure 7 shows the results of evaluating the H2O2 sensitivity of outdoor plants and relQ gene deletion mutants (**P < 0.01, ***P < 0.001).

[0020] Figure 8 shows the results of evaluating cell internalization of outdoor strains and relQ gene deletion mutant strains (***P < 0.001).

[0021] Figure 9 shows the results of evaluating the phagocytosis patterns of outdoor strains and relQ gene deletion mutant strains (**P < 0.05, ***P < 0.001).

[0022] Figure 10 is a diagram showing the results of evaluating the efficacy of the vaccine composition of the present invention in target animals.

[0023] Figure 11 is a diagram showing the results of evaluating the efficacy of the vaccine composition of the present invention in target animals.

[0024] The present invention is described in detail below.

[0025] The present invention provides a mutant strain of Escherichia coli or Staphylococcus aureus caused by bovine mastitis.

[0026] The Escherichia coli of the present invention is Escherichia coli (E. coli), a Gram-negative bacterium. This E. coli is known as a representative causative agent of environmental mastitis, invading mammary tissue through various environmental factors such as soil, feces, genital secretions, litter, udders and nipples, and contaminated milking machine wash water, causing opportunistic infections. This has been reported to cause temporary clinical mastitis.

[0027] The Staphylococcus aureus of the present invention is Staphylococcus aureus (S. aureus), commonly referred to as "Staphylococcus aureus" or "Staphylococcus aureus," and is a type of staphylococcus belonging to the Gram-positive bacteria. Staphylococcus aureus is a representative causative agent of infectious mastitis, infecting the mammary glands and transmitting it to other dairy cows through the milking process. As a result, it is known to cause persistent subclinical or chronic mastitis in dairy cows.

[0028] Bovine mastitis is the most common bacterial disease in dairy cows, and its causative agents are generally divided into infectious and environmental agents. The main causative agents of infectious mastitis include Staphylococcus aureus, Streptococcus dysgalactiae, and Streptococcus dysgalactiae, and they are mainly transmitted during the milking process through contaminated milking machines, milkers' hands, cleaning sponges, and towels. The main causative agents of environmental mastitis include Escherichia coli, Streptococcus uberis, and coagulase-negative staphylococci (CNS), and they mainly enter the udder through teats that have come into contact with a fecal-contaminated environment.

[0029] The Escherichia coli or Staphylococcus aureus of the present invention may be a strain isolated from a dairy cow infected with clinical or subclinical mastitis. Clinical mastitis is a condition in which sediment or foreign substances are visually visible in raw milk, and the udder becomes swollen, feverish, painful, and causes breast dysfunction. Severe acute mastitis is accompanied by systemic high fever, depression, chills, loss of appetite, and rapid weight loss. Acute mastitis shows typical inflammatory symptoms such as swelling, redness, and pain. Subacute mastitis is classified as a type similar to acute mastitis but without distinct systemic symptoms. On the other hand, subclinical mastitis is a condition that cannot be identified with the naked eye, and can be identified through field or laboratory diagnostic methods such as the California Mastitis Test (CMT) or a milk bacterial culture test. The incidence is 15 to 40 times higher than clinical mastitis, and in most cases, it progresses to clinical mastitis over time. Chronic mastitis is a condition in which the inflammation persists for several months or continues until the next lactation, and bacteria may remain dormant in the breast and recur due to factors such as stress or deteriorating health.

[0030] The present inventors isolated a highly pathogenic E. coli strain without antibiotic resistance from a domestic dairy cow infected with mastitis. Subsequently, they generated a gene-inactivation mutant strain by inserting antibiotic resistance genes into the relA and spoT genes of the E. coli strain, thereby inactivating the function of these genes. Furthermore, they isolated ST72 and ST5 Staphylococcus aureus strains from a domestic dairy cow infected with mastitis, and generated mutant strains lacking the relQ gene, thereby obtaining an attenuated staphylococcal mutant strain.

[0031] Accordingly, the present invention provides an E. coli mutant strain in which an antibiotic resistance gene is inserted into the relA and spoT genes, thereby inactivating the function of the corresponding gene.

[0032] The relA (RelA / SpoT homologous protein) and spoT (Stringent starvation protein) genes are key genes involved in the energy stress response of bacteria. The relA gene encodes an enzyme that synthesizes (p)ppGpp (guanosine tetraphosphate or pentaphosphate) under stress conditions, and the spoT gene regulates intracellular (p)ppGpp metabolism together with relA by performing both the synthesis and degradation of (p)ppGpp.

[0033] The E. coli mutant strain of the present invention may preferably have a kanamycin resistance gene inserted into the relA gene and a chloramphenicol resistance gene inserted into the spoT gene. The Staphylococcus aureus mutant strain may be one deposited under the accession number KCCM13583P or KCCM13584P.

[0034] In addition, the present invention provides a Staphylococcus aureus mutant strain lacking the relQ gene.

[0035] The above relQ gene is known to be a gene encoding an enzyme that synthesizes (p)ppGpp.

[0036] The Staphylococcus aureus mutant strain of the present invention may preferably be a strain in which 468 bp (base pairs) of the relQ gene are deleted. The relQ gene and the gene in which the 468 bp base pairs are deleted are represented by SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The Staphylococcus aureus mutant strain may be a strain deposited under the accession number KCCM13583P or KCCM13584P.

[0037] The vaccine composition comprising the E. coli mutant strain and the Staphylococcus aureus mutant strain according to the present invention exhibits excellent immunogenicity, thereby providing an effect capable of effectively preventing mastitis infection in dairy cows.

[0038] Accordingly, the present invention provides a vaccine composition for preventing mastitis in dairy cows comprising the above-described E. coli mutant strain or Staphylococcus aureus mutant strain.

[0039] The term “prevention” as used herein means any action that suppresses or delays the onset of a disease by administering a composition.

[0040] The term "vaccine" as used herein refers to a composition containing an antigen used to induce immunity in humans or animals for the purpose of preventing infectious diseases. The vaccine of the present invention may include a live attenuated vaccine, an inactivated vaccine, a killed vaccine, a subunit vaccine, a synthetic vaccine, a genetically engineered vaccine, etc., but an inactivated vaccine is preferred. Live vaccines carry concerns about adverse effects, such as pathogenic reversion, and therefore, inactivated vaccines are preferred for safer vaccination.

[0041] To manufacture an inactivated vaccine, a step of inactivating the mutant strain according to the present invention is required. The strain can be inactivated using conventional methods such as formalin treatment or heat treatment. Inactivation is preferably performed using formalin treatment, but is not limited thereto.

[0042] The above vaccine composition may be interpreted as having the equivalent meaning of "immunogenic composition" or "immunological composition." The immunological composition refers to a substance comprising one or more antigens capable of inducing a cellular immune and / or humoral immune response in a host. Typically, an "immunological response" includes, but is not limited to, one or more of biological responses such as the production of antigen-specific antibodies, activation or production of B cells, helper T cells, suppressor T cells, cytotoxic T cells, and gamma-delta T cells. Preferably, the host (cow) exhibits a therapeutic or protective immune response, thereby increasing resistance to new infections or reducing the clinical severity of the disease. Such protective effects may be demonstrated by a reduction in clinical symptoms in the infected host, a shortened recovery time, a reduction in the number of bacteria in infected tissues, etc.

[0043] The vaccine composition of the present invention may further comprise a veterinarily acceptable carrier, diluent, and adjuvant. The carrier may be water-soluble or water-insoluble, and water-soluble carriers may include physiological saline, buffer solutions, aqueous emulsions or suspensions, and non-aqueous carriers may include, but are not limited to, propylene glycol, polyethylene glycol, edible oils (e.g., olive oil), ethyl oleate, and the like. The diluent refers to a substance used to adjust the concentration of the vaccine to an appropriate level or to improve the physical stability and injectability of the composition. The diluent is usually a non-toxic and physiologically acceptable solution, and may include, but is not limited to, physiological saline (0.9% NaCl), phosphate-buffered saline (PBS), sterile distilled water, a sugar solution (e.g., 5% dextrose solution), a sugar-electrolyte mixture solution (e.g., Ringer's solution, Ringer-dextrose), and the like. The above adjuvant is a substance that promotes an immune response to an antigen and can be administered together with the composition of the present invention to enhance the immune effect. These adjuvants can be administered simultaneously or sequentially with time intervals. Available adjuvants include, but are not limited to, ISA70, aluminum hydroxide gel (Alum), Freund's incomplete or complete adjuvant, aluminum hydroxide, and vegetable or mineral oils.

[0044] The vaccine composition of the present invention can be administered in an immunologically effective amount. The term "immunologically effective amount" means an amount sufficient to induce a disease prevention effect and not to cause an excessive immune response. The dosage can be appropriately adjusted by those skilled in the art according to the antigen characteristics, body weight, health condition, age, sex, sensitivity, administration route, etc. of the target animal, and can be administered once or multiple times. The effective bacterial count content in the vaccine composition of the present invention is 106 10 inland 9 cfu, preferably 10 7 10 inland 8 It could be CFU.

[0045] The above vaccine composition may be prepared as an oral or parenteral formulation, and is preferably prepared as an injection, which is a parenteral formulation. Administration routes include transdermal, intramuscular, intraperitoneal, intradermal, subcutaneous, and intranasal routes, with intramuscular injection being particularly preferred but not limited thereto.

[0046] The vaccine of the present invention can be administered directly, either as a mutant strain itself or in combination with a carrier, or can be administered orally by mixing it with feed or drinking water. Proteins, sugars, aqueous solutions, suspensions, emulsions, and the like can be used as carriers. Any substance known to those skilled in the art may be included without particular limitation.

[0047] In addition, the present invention provides a method for preventing bovine mastitis, comprising administering to a dairy cow a vaccine composition comprising a mutant strain of E. coli or a mutant strain of Staphylococcus aureus.

[0048] The method for preventing mastitis of the present invention aims to induce and increase an immune response in a living body by administering the mutant strain, and more specifically, to reduce the incidence of the disease and alleviate clinical severity by regulating the expression of factors related to antibody production and immune response.

[0049] The present invention provides a method for producing a vaccine composition, comprising the steps of culturing an E. coli mutant strain or a Staphylococcus aureus mutant strain according to the present invention; and the step of inactivating the mutant strain.

[0050] In addition, the present invention provides a multivalent vaccine composition for preventing infection with E. coli and Staphylococcus aureus in dairy cows, comprising an E. coli mutant strain in which an antibiotic resistance gene is inserted into the relA and spoT genes to inactivate the function of the corresponding gene; and a Staphylococcus aureus mutant strain in which the relQ gene is deleted.

[0051] The vaccine composition of the present invention can be applied as a monovalent vaccine for preventing E. coli or Staphylococcus aureus infections by utilizing each mutant strain individually. Furthermore, it can also be administered as a multivalent vaccine by including all mutant strains. This allows for the simultaneous induction of immune responses against E. coli and Staphylococcus aureus, the major causative agents of bovine mastitis, thereby enabling the implementation of an efficient multivalent vaccine strategy.

[0052] Duplicate content is omitted in consideration of the complexity of this specification, and terms not otherwise defined in this specification should be understood to have the meanings commonly used in the technical field to which the present invention belongs.

[0053] Hereinafter, the present invention will be described in more detail through examples. However, these examples are intended to more clearly illustrate the content of the present invention and are not intended to limit the spirit or scope of the present invention. Those skilled in the art will readily appreciate that various modifications and applications are possible without departing from the technical spirit and scope of the present invention.

[0054] <Example 1> Selection and Characterization of Candidate E. coli Strains for Bovine Mastitis Vaccine

[0055] We analyzed E. coli strains originating from domestic cow mastitis, selected highly pathogenic candidate strains, and ultimately determined seed strains for vaccine development.

[0056] <1-1> Selection of candidate strains through pathogenicity evaluation of E. coli caused by mastitis in dairy cows

[0057] Phylogenetic analysis and pathogenicity factor investigation were performed on 98 Escherichia coli strains isolated from clinical mastitis in domestic dairy farms. Highly pathogenic strains A and B1 were found to be the majority, and representative strains belonging to ST10, ST58, and ST906 were selected. As a result of comparative pathogenicity evaluation using a mouse infection model, the EC53 strain belonging to ST10, which did not show antibiotic resistance, was finally selected as the seed strain for the vaccine of the present invention.

[0058] <1-2> Production of attenuated E. coli mutant strains

[0059] A double-inactivation mutant (relA-spoT mutant) was constructed by inserting antibiotic resistance genes into the relA and spoT genes of the selected highly pathogenic EC53 strain (Fig. 1). The insertion of these genes was performed using targeted mutagenesis, and the accuracy of the insertion site was confirmed through sequencing.

[0060] EC53 wild-type (WT) strain and relAspoT mutant strain were cultured under the same conditions, and CFU (Colony Forming Unit) and OD were measured at each time point. 600 nm (turbidity) was measured.

[0061] As a result, as shown in Fig. 2, the relAspoT mutant strain showed a somewhat reduced growth rate for the first 8 hours of culture, which is interpreted as an indicator of the attenuation effect.

[0062] The above relAspoT mutant strain was named Escherichia coliTYH-EC1.2 and deposited with the Korea Microbiological Conservation Center under the accession number KCCM13582P.

[0063] <1-3> Attenuation analysis of E. coli vaccine mutant strains

[0064] The reduction in pathogenicity of the above relAspoT mutant strain was evaluated through cell adhesion assay.

[0065] Specifically, a comparative experiment was conducted on cell adhesion of the wildtype EC53 strain and the relAspoT mutant to MDBK cells (bovine kidney epithelial cell line). Each strain was treated with MDBK cells under identical conditions, and non-adherent strains were removed through washing. The colony forming units (CFU) of the attached strains were measured.

[0066] As a result, as shown in Fig. 3, it was confirmed that the cell adhesion ability of the relAspoT mutant was significantly reduced compared to the wildtype strain (three independent experiments, p < 0.05).

[0067] The above results indicate that the E. coli mutant strain of the present invention is an attenuated strain with reduced pathogenicity and cell adhesion ability, and that it can contribute to ensuring safety and effectiveness as a vaccine strain.

[0068] <Example 2> Selection and characterization of Staphylococcus aureus candidate strains for cow mastitis vaccine

[0069] Through isolation and genetic characterization of Staphylococcus spp. caused by domestic cow mastitis, highly pathogenic strains were selected and genetically defective mutant strains were created for vaccine development.

[0070] <2-1> Isolation and genotypic analysis of Staphylococcus aureus caused by mastitis in dairy cows

[0071] A total of 135 staphylococcal isolates were isolated from clinical and subclinical mastitis cases on domestic dairy farms, and approximately 84% of these were identified as Staphylococcus aureus (S. aureus). Molecular genetic analysis revealed that the ST188 genotype accounted for the highest proportion (approximately 40%), and antibiotic susceptibility testing confirmed resistance to β-lactam antibiotics in some S. aureus and related strains.

[0072] <2-2> Production of Staphylococcus aureus mutant strains

[0073] Using the homologous recombination technique, we constructed mutant strains lacking the relQ gene for Staphylococcus aureus ST5 and ST72 strains, which were selected as the main target strains for vaccine development (Fig. 4). To this end, we constructed a knockout vector based on pCL10, a Staphylococcus aureus-specific knockout plasmid. Whole genome sequencing (WGS) of the constructed mutants confirmed a 468 bp deletion in the relQ gene, verifying complete inactivation of the gene. In particular, we successfully constructed relQ-deleted mutants (ST5 MRSA relQ mutant, ST72 MSSA relQ mutant) from strains with the ST5 MRSA and ST72 MSSA genotypes, respectively. The above ST5 MRSA relQ mutant strain and ST72 MSSA relQ mutant strain were named Staphylococcus aureusTYH-SA1.1 and Staphylococcus aureusTYH-SA2.1, respectively, and deposited with the Korea Microbiological Conservation Center under the accession numbers KCCM13583P and KCCM13584P.

[0074] <2-3> Evaluation of growth ability of Staphylococcus aureus mutant strains

[0075] To evaluate the growth ability of the wild-type strain and relQ-deficient mutant, CFU and OD 600The survival and growth rates were compared by measuring nm.

[0076] As a result, as shown in Fig. 5, in the case of ST5 MRSArelQmutant, the growth rate was somewhat reduced from immediately after culture to about 12 hours compared to the wild-type strain, but it showed a growth level similar to that of the wild-type strain after the stationary phase. On the other hand, no difference in growth rate was observed for ST72 MSSArelQmutant compared to the wild-type strain.

[0077] <2-4> Antimicrobial peptide (cathelicidins) and H2O2 susceptibility evaluation

[0078] The viability of relQ deletion mutants was evaluated using antimicrobial peptides derived from bovine (BMAP-28), human (LL-37), and chicken (CATH-2).

[0079] As a result, as shown in Fig. 6, ST5 MRSArelQmutant showed higher viability than wild-type at 2 hours after peptide treatment, but after 4 hours of treatment, viability against BMAP-28 and CATH-2 significantly decreased. On the other hand, ST72 MSSArelQmutant showed viability similar to wild-type at 2 hours, and after 4 hours, viability against BMAP-28 and CATH-2 actually increased.

[0080] To determine the resistance pattern to oxidative stress, one of the host defense factors, for relQ-deficient mutants, a sensitivity evaluation to H2O2 was performed.

[0081] In addition, as shown in Fig. 7, when treated with H2O2 to evaluate sensitivity to oxidative stress, the relQ defective mutant strain showed significantly increased sensitivity to H2O2 compared to the ST5 MRSA and ST72 MSSA wildtype strains.

[0082] <2-5> Evaluation of host cell penetration ability (cell internalization)

[0083] The number of strains that penetrated into the cells was measured after 2 hours of co-culture under MOI conditions of 1:1, 1:10, and 1:100 using the MDBK cell line.

[0084] As a result, as shown in Fig. 8, in the case of ST5 MRSA, the relQ deletion mutant showed an overall decrease in cell penetration ability compared to the wild-type strain, and a significant decrease was confirmed under MOI 1:10 and 1:100 conditions. The ST72 MSSA relQ mutant also showed a significantly lower cell penetration ability than the wild-type strain under MOI 1:1 and 1:10 conditions.

[0085] <Example 3> Analysis of phagocytosis patterns of Staphylococcus aureus vaccine candidate strains

[0086] To evaluate the degree of cellular immune response induction, phagocytosis of macrophages was analyzed using mouse-derived bone marrow macrophages (BMDM).

[0087] As a result, as shown in Fig. 9, in the early stage of the reaction (1 hour), the phagocytosis rate by macrophages increased in the relQ-deficient mutant strain compared to the ST5 MRSA and ST72 MSSA wild-type strains, but after 24 hours of the reaction, the phagocytosis by BMDM tended to decrease significantly.

[0088] This suggests its safety as a vaccine candidate strain and its potential for immunomodulation, as it can suppress the secretion of inflammatory cytokines by macrophages and consequently reduce excessive inflammatory responses.

[0089] <Example 4> Production of inactivated vaccine using cow mastitis vaccine candidate strain

[0090] The E. coli mutant strain of Example 1 and the Staphylococcus aureus mutant strain of Example 2 were inactivated by formalin treatment, and a vaccine composition was prepared using them as an antigen.

[0091] Specifically, the mutant strains were cultured in Luria-Bertani (Difco) or Trypticase Soy broth (Difco) medium, and the optical density at 600 nm (OD 600 After culturing until the chromatin (nm) reached 0.6, the cells were collected and washed three times with sterile phosphate buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, pH 7.2) by centrifugation at 8000×g for 15 min at 4°C. The washed cells were mixed with PBS (pH 7.2) containing 0.4% formalin (MERCK, Germany), shaken for 1 h at 37°C, and left to stand for 18 h at 4°C. Afterwards, the cells killed by formalin were collected by centrifugation at 12000×g for 30 min at 4°C, washed with sterile PBS, and finally lysed at 10 8 The cells were resuspended in PBS to obtain colony-forming units (CFU). Sterility testing of formalin-inactivated cells was performed in Luria-Bertani (Difco) or Trypticase Soy broth (Difco) media, and the cells were cultured at 37°C for 7 days, and the presence of contamination was observed.

[0092] <Example 5> Evaluation of vaccine efficacy and safety using target animals (dairy cows)

[0093] The efficacy and safety of the vaccine manufactured in Example 4 were evaluated in dairy cows, the target animals.

[0094] Specifically, the experiment was conducted at the Kangwon National University ranch, and five heifers (first-time mothers, full-term) were brought in and underwent a five-week acclimatization process before the experiment. All test cows were managed according to the breeding method of the test ranch. If there were any abnormalities in the udder shape such as blind bundles, udder ptosis, or nipple damage before the experiment, if there was a systemic disease or mastitis that could affect the test, if antibiotics were administered to treat clinical mastitis within 28 days of the start of the test, or if NSAIDs were administered 3 days before the test, they were excluded from the test group. In addition, all dairy cows were raised under the same feeding and management conditions during the experimental period, and the owner or a veterinarian observed and recorded their health status (behavior, attitude, udder condition, etc.) every day.

[0095] Finally, five dairy cows were administered 1) saline (MOCK control group), 2) a commercially available vaccine from another company (positive control group), 3) EC53 relAspoT mutant (E. coli vaccine), 4) ST72 MSSA mutant (S. aureus vaccine), and 5) EC53 relAspoT mutant + ST72 MSSA mutant (E. coli vaccine + S. aureus vaccine). Immune responses were observed for three weeks after the first vaccination, and then the immune responses were checked for three weeks in the same manner after the second vaccination.

[0096] No local adverse reactions (pain, redness, swelling, fever, etc.) or systemic adverse reactions (decreased appetite, elevated body temperature, shock, etc.) were observed in any animal after vaccination. In addition, the somatic cell count in milk collected after parturition ranged from 12 to 75 (×10³ cells / mL), indicating no abnormal immune response to the vaccine.

[0097] Serum antibody titer analysis was performed by ELISA using jugular blood collected weekly. Antigens were prepared by culturing E. coli EC53 wildtype and S. aureus ST72 wildtype strains, respectively, and lysing the cells with a French pressure cell press after treatment with lysostaphin or lysozyme. After centrifugation, the supernatant was filtered through a 0.2 μm filter to prepare an antigen solution, which was quantified using a BCA protein assay and diluted to 40 μg / mL. The antigen was coated on a 96-well plate at 1 μg / well, and absorbance (OD) was determined through blocking, serum reaction, HRP-conjugated antibody reaction, and TMB substrate reaction. 450 ) was measured to evaluate antibody titer.

[0098] As a result, as shown in Fig. 10, cows administered the EC53 relAspoT mutant strain (E. coli vaccine) and ST72 MSSA mutant strain (S. aureus vaccine) showed higher antibody titers against E. coli and Staphylococcus aureus, which are causative agents of mastitis, compared to the control group and positive control group, starting 3 weeks after vaccination.

[0099] In addition, a reproducibility test was performed on antibody formation ability using 10 dairy cows. 1) 2 cows were treated with saline (MOCK control group), 2) 2 cows were treated with a commercially available vaccine (positive control group), 3) 3 cows were treated with an EC53 relAspoT mutant strain (E. coli vaccine), and 4) 3 cows were treated with an ST72 MSSA mutant strain (S. aureus vaccine). The reproducibility test was performed as described above, and the results were analyzed.

[0100] As a result, as shown in Fig. 11, all dairy cows administered the EC53 relAspoT mutant strain (E. coli vaccine) and ST72 MSSA mutant strain (S. aureus vaccine) showed higher antibody titers against E. coli and Staphylococcus aureus, which are causative agents of mastitis, compared to the control group and positive control group from 3 weeks after vaccination.

[0101] [Accession number]

[0102] Name of depositor: Korea Center for Microbiological Conservation (KCCM)

[0103] Accession number: KCCM13582P

[0104] Date of acceptance: 20250707

[0105]

[0106] Name of depositor: Korea Center for Microbiological Conservation (KCCM)

[0107] Accession number: KCCM13583P

[0108] Date of acceptance: 20250707

[0109]

[0110] Name of depositor: Korea Center for Microbiological Conservation (KCCM)

[0111] Accession number: KCCM13584P

[0112] Date of acceptance: 20250707

[0113]

[0114] [Correction pursuant to Rule 91, 04.08.2025]

[0115] [Correction pursuant to Rule 91, 04.08.2025]

[0116] [Correction pursuant to Rule 91, 04.08.2025]

Claims

1. As a mutant strain of Escherichia coli or Staphylococcus aureus caused by cow mastitis, The above E. coli has antibiotic resistance genes inserted into the relA and spoT genes, and the function of the genes is inactivated. The above-mentioned Staphylococcus aureus is a mutant strain characterized by a deletion of the relQ gene.

2. In paragraph 1, The above E. coli is a mutant strain, characterized by being deposited under the accession number KCCM13582P.

3. In paragraph 1, The above-mentioned Staphylococcus aureus is a mutant strain, characterized in that it has been deposited under the deposit number KCCM13583P or KCCM13584P.

4. A vaccine composition for preventing mastitis in cows, comprising a mutant strain of any one of claims 1 to 3.

5. In paragraph 4, A vaccine composition characterized in that the above mutant strain is inactivated.

6. In paragraph 4, A vaccine composition characterized in that the above inactivation is by formalin treatment or heat treatment.

7. In paragraph 4, A vaccine composition characterized in that the vaccine composition is administered by at least one route selected from the group consisting of oral, transdermal, intramuscular, intraperitoneal, intradermal, subcutaneous, and nasal routes.

8. In paragraph 4, A vaccine composition characterized in that the vaccine composition further comprises at least one selected from the group consisting of a carrier, a diluent, and an adjuvant.

9. A method for preventing bovine mastitis, comprising administering to a dairy cow a vaccine composition according to paragraph 4. 10.(a) A step of culturing a mutant strain according to paragraph 1; and (b) A method for producing a vaccine composition, comprising a step of inactivating the mutant strain.

11. E. coli mutant strain in which antibiotic resistance genes are inserted into the relA and spoT genes, thereby inactivating the function of the genes; and A multivalent vaccine composition for preventing bovine mastitis, comprising a Staphylococcus aureus mutant strain lacking the relQ gene.

12. In paragraph 11, The above E. coli is deposited under the deposit number KCCM13582P. A multivalent vaccine composition, characterized in that the above staphylococcus is deposited under the deposit number KCCM13583P or KCCM13584P.

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