KR101013226B1 - Vaccine for preventing canine viral diarrhea - Google Patents

Abstract

The present invention provides at least one selected from the group consisting of inactivated canine parvovirus (CPV), inactivated canine coronavirus (CCoV) and inactivated canine rotavirus (CRV). It relates to a vaccine for the prevention of canine viral diarrhea containing. The vaccine of the present invention is a useful vaccine that can prevent all of the viral viral diarrheal diseases caused by CPV, CRV and CCoV at the same time in a single inoculation, and minimize the damage caused by diarrheal diseases in dogs with excellent safety.

Dog, diarrhea, vaccine, parvovirus, rotavirus, coronavirus

Description

Vaccine for preventing canine viral diarrhea

The present invention relates to a vaccine for the prevention of canine viral diarrhea, and more particularly, to canine parvovirus (CPV), canine rotavirus (CRV), and canine coronavirus (CCoV). The present invention relates to a vaccine for preventing canine viral diarrhea containing at least one selected from the group consisting of

Representative causes of canine viral diarrhea include parvovirus, coronavirus and rotavirus.

Canine parvovirus has been reported worldwide since 1987, and it was confirmed that it was the first epidemic in Gyeonggi-do in Korea in the 1980s. In addition, in the case of canine parvovirus, a mutant virus exists, and representative viruses are CPV-2a and CPV-2b. Citing recent presentations at the 2004 Fall Conference of the Korean Veterinary Society, 36 parvoviruses were detected in 39 diarrhea fecal samples, including 3 CPV-2a (8.3%) and 20 CPV-2b infections ( 55.5%) and 13 mixed infections (36.1%) of CPV-2a and CPV-2b were reported. As a result of investigating the immunogenicity of the vaccine using CPV-2b, it is reported that the vaccine using CPV-2b has better protective antibodies against CPV-2 and CPV-2b than the vaccine using CPV-2 as an antigen. Pratelli et al . , Clin . Diag , Lab , Immunol , 2001). According to a recent thesis from Seoul National University College of Veterinary Medicine, 51 cases of dog diarrhea were positive for parvovirus. Among them, 49 cases of CPV-2a (96.0%) and 1 case of CPV-2b (2%) One case (2%) of CPV-2a mutants was reported. Cross-immunogenicity of CVP-2 and the three mutants, CPV-2a, CPV-2b, and CPV-2a mutants, showed that CPV-2, which is currently used as a vaccine strain, has low cross reactivity against three mutants. Cross-reactivity was reported to be similar among the mutants in dogs (Kang et al . , Virus Genes, 2008).

Canine coronaviruses cause gastrointestinal infectious diseases, including vomiting, diarrhea and dehydration, and infections in dogs and in dogs, but especially in dogs and are susceptible to breeds and ages, with rapid spread and high morbidity. In particular, it is spread within a short time in a breed dog. Canine coronavirus is considered to be an important cause of digestive diseases of dogs because it is mixed with canine parvovirus and worsens symptoms.

Canine rotavirus does not show severe clinical symptoms, but can cause problems with enteritis accompanied by lethargy or vomiting in young children younger than 2 weeks. Although recent reports suggest that dog rotavirus is less common, dog rotavirus has been reported in dogs with severe diarrhea in Japan (Mochizuki et al . , J. Vet). Med Sci , 2001), reports also show that dogs have a high rate of antibody positive rates. In case of susceptibility to viral enteritis, susceptible dogs have 100% infection, and the mortality rate is 25%.

In other words, the virus that causes viral diarrhea in dogs is not only diverse, but also frequently infected with paronaviruses. In particular, when combined infection is higher than when infected alone, the mortality rate is higher, and when the infants of young age are infected, the mortality rate is higher. Therefore, a vaccine that can sufficiently prevent CPV-2a, CPV-2b, CRV and CCoV, which are the main causes of viral viral diarrheal disease, is minimized the damage caused by viral diarrheal disease in dogs. This is necessary.

The present invention can sufficiently prevent CPV-2a, CPV-2b, CRV and CCoV, which are the main causes of viral viral diarrheal disease, by minimizing the damage caused by diarrheal disease in dogs with excellent safety. It is to provide a vaccine.

In order to solve the above problems, the present invention is a vaccine for preventing viral diarrhea of dogs containing at least one selected from the group consisting of inactivated CPV-2a and CPV-2b, inactivated CRV and inactivated CCoV and It provides a manufacturing method.

Hereinafter, the vaccine of the present invention will be described in detail.

The vaccine of the present invention contains one or more selected from the group consisting of inactivated CPV-2a and CPV-2b, inactivated CCoV and inactivated CRV. The vaccine of the present invention is a canine parvovirus, and may include both inactivated CPV-2a and CPV-2b, or may include any one of inactivated CPV-2a and CPV-2b. When both inactivated CPV-2a and CPV-2b are included in the vaccine of the present invention, the inactivated CPV-2a and CPV-2b are contained in a 1: 1 ratio.

The vaccine of the present invention is preferably a combination vaccine containing both inactivated CPV-2a and CPV-2b, inactivated CRV and inactivated CCoV. Inactivated CPV-2a and CPV-2b, inactivated CRV, and inactivated CCoV in the combination vaccine of the present invention are contained at a ratio of 40:25:25 or 30:30:30. Preferably, the CPV-2a, CPV-2b, CRV and CCoV are contained in a 1: 1: 1 ratio. CPV-2a and CPV-2b are contained in a 1: 1 ratio.

The present invention provides a vaccine in which an immunoadjuvant is added to the vaccine. As an adjuvant, a conventional adjuvant known to increase the dog's immunity when added to the vaccine can be used. Preferably, IMS1312N or Montanide Gel, RehydraGel may be used in the IMS series of Seppic (France) as an immunopotentiator. The amount of adjuvant added depends on the titer of the virus, but is preferably added at 10-15% (w / w).

Suitable preservatives are thimerosal, formaldehyde, gentamicin.

Preferably, the vaccine of the present invention is CRV passaged in the CPV-2a and CPV-2b, CCoV culture and MA104 cells (monkey kidney cells), the antigen used in the vaccine preparation passaged in CRFK (Crandell Feline Kidney) cells It is a vaccine that inactivates each virus in culture.

Preferably, the present invention is a vaccine comprising a supernatant obtained by lyolysis and centrifugation when 60-90%, preferably 70% or more of cell degeneration effects are obtained after inoculating each cell with a virus. .

CPV-2a and CPV-2b used in the vaccine of the present invention were deposited in the Korean Collection for Type Cultures (KCTC) of the Korea Institute of Biotechnology, an international depository, respectively, CPV-2a is KCTC 11082BP, and CPV-2b is KCTC 11083BP. You have been assigned a deposit number. In addition, the CRV used in the vaccine of the present invention was assigned an accession number of KCTC 11081BP, CCoV was given an accession number of KCTC 11317BP.

The vaccine of the present invention can be administered by oral or intramuscular vaccination. The vaccine of the present invention contains CPV-2a, CPV-2b, CCoV, and CRV at 10 ^6.0 -10 ^7.0 TCID ₅₀ / ml, and the puppy is first inoculated at 4-5 weeks of age and 2 at 6-7 weeks of age. Inoculate tea at 1.0 ml per horse, inoculate 1st 4-5 weeks before delivery, 2nd inoculation 2-3 weeks before delivery, and 2.0ml per horse, and 2-3 weeks apart for small dogs. Two doses of 1.0 ml per horse are inoculated. For large dogs, two doses of 2.0 ml per horse are given 2-3 weeks apart.

In the mixed vaccine of the present invention, when compared with the group administered with the vaccine alone containing CPV-2b, CCoV or CRV, respectively, as shown in FIGS. 4 to 6, 2 weeks after the second inoculation The antibody titers measured were able to induce similar levels. Therefore, the combined vaccine of the present invention can prevent all of the dog viral diarrheal diseases caused by CPV, CRV and CCoV at the same time in a single inoculation.

The combination vaccine of the present invention showed no incidence of intravenous diarrhea in the farm when the safety was verified in the field test, and there was no significant difference in clinical symptoms and mortality in the inoculated group compared to the control group (Tables 7 to 9). Reference). Thus, the vaccine of the present invention can be used for the prevention of canine viral diarrhea without distrust of the vaccine.

Hereinafter, the vaccine manufacturing process of the present invention will be described in detail.

1.Isolation and Culture of CPV-2a and CPV-2b, CRV and CCoV

In order to obtain the virus to be used as the antigen of the vaccine of the present invention, CPV-2a, CPV-2b, CRV and CCoV are isolated from the specimens of dogs with outdoor diarrhea, and each virus is propagated through passage culture.

CPV-2a, CPV-2b, CRV and CCoV were first isolated from specimens of dogs with outdoor diarrhea. The presence or absence of these was used by rapid diagnostic kit and PCR (polymerase chain reaction).

For passage of each virus, CRFK cells are preferably used for CPV-2a, CPV-2b, and CCoV, and MA104 cells for CRV. Each of the viruses cultivated using the cells can be usefully used for antigen presentation with excellent antibody formation ability. The passage is continued until cell degeneration effects are achieved while maintaining both proliferation and immunogenicity. Cytopathic forms of each virus are shown in FIGS.

The culture obtained is passaged preferably until 60-90%, preferably 70% or more of cell degeneration effect is obtained to obtain each virus. In the case of CPV-2a and CPV-2b, it was passaged up to 50 generations, in the case of CCoV passages up to 30 generations, it is preferable to passage up to 25 generations in the case of CRV. After passage passage up to a certain passage is completed, the obtained culture solution is lyophilized and the supernatant obtained after centrifugation is separated. The separated upper layer contains each of the viruses. In this case, the virus titer of CPV-2b was higher than HAG 2 ¹¹ and CCoV and CRV of 10 ^6.5 TCID ₅₀ /0.1ml, respectively.

2. Inactivation of the virus

The virus in the supernatant is inactivated by conventional methods, for example by inactivating agent formalin or bromoethylamine hydrobromide solution (BEI). The concentration at which the inactivating agent is added may vary depending on the titer of each of the viruses contained in the supernatant. For example, when inactivated with formalin, it is preferred to add at a concentration of 0.2-0.5%.

After inactivation, in order to neutralize the used inactivating agent, 1M sodium thiosulfate is added and neutralized by slowly stirring at 37 ° C for 1 hour and 4 ° C overnight.

3. Preparation of Inactivated Combination Vaccines

The mixed vaccine according to the present invention comprises mixing a culture solution of the inactivated virus. Although the ratio of mixing the culture solution is not limited, it is preferable to mix in a ratio of 1: 1: 1. In addition, immunosuppressants such as IMS1312N (Seppic, France) or RehydraGel may be added to enhance the dog's immunity.

The mixed vaccine of the present invention can prevent all of the dog viral diarrheal diseases caused by CPV, CRV and CCoV at the same time in one shot. In addition, the present invention can provide a useful vaccine by minimizing the damage caused by diarrhea in dogs while excellent in safety.

Hereinafter, the present invention will be described in detail by way of examples. However, the present invention is not limited by the following examples.

Example : Preparation of Mixed Vaccine

(1) Isolation and culture of vaccine strains for use in combination vaccines

Detects CPV-2a, CPV-2b, CRV, and CCoV in poultry affected by outdoor diarrhea. Use a quick diagnostic kit (Anigen) or the commonly known PCR method. Sterile PBS is added to the specimens that are positive for each virus to make 10% emulsion. After digestion with trypsin solution (Trypsin EDTA, Gibco, Cat. No. 154--054), the dispersed CRFK cells and MA-104 cells were suspended in proliferation medium, subdivided into culture vessels and then 2-3 days at 37 ° C. Were cultured for 3 days and used for subculture after 3 days of culture. The medium for CRFK cell proliferation consists of 950 ml of D-MEM, 50 ml of FBS (fetal serum albumin), an appropriate amount of antimycotics and 1% of 7% sodium bicarbonate solution, adjusted to pH 7.0-7.4, and MA-104 cells were α- Consists of 950 ml of MEM, 50 ml of FBS, titration of antimycotics and 1% of a 7% sodium bicarbonate solution, adjusted to pH 7.0-7.4. In the case of CPV-2a and CPV-2b, the cultured CRFK cells were inoculated to be 5% of the total medium volume and the CRFK cell proliferation medium was added and cultured at 37 ° C. for 3-5 days. Subcultures were carried out until the effect of cell degeneration was 70% or more, and the obtained culture was aseptically lyophilized and stored at -80 ° C. In the case of CCoV, inoculated with 1.0 moi of CRFK cells in which monolayers were formed at 80% or more, and then the CRFK cell proliferation medium was added thereto and cultured at 37 ° C. for 3-5 days. Subcultures were carried out until the effect of cell degeneration was 70% or more, and the obtained culture was aseptically lyophilized and stored at -80 ° C. In case of CRV, inoculate 1.0 moi into MA-104 cells in which 80% or more monolayers were formed, and then use a cell growth medium (α-MEM 950 ml, trypsin 5 µg / ml, antimycotics titration amount and 7% sodium bicarbonate solution 1%). It was configured and pH 7.0-7.4) was put and incubated for 3-5 days at 37 ℃. Subcultures were carried out until the effect of cell degeneration was 70% or more, and the obtained culture was aseptically lyophilized and stored at -80 ° C. CPV-2a and CPV-2b were cultured to 50s, and the virus titer was 2 ¹⁴ (HAU) by the hemagglutination inhibition test. CCoV was subcultured to 30 generations, and the antibody titer was 10 ^6.5 TCID ₅₀ /0.1 ml according to the virus serum neutralization test. The CRV was passaged up to 25 generations, and the antibody titer was 10 ^6.5 TCID ₅₀ /0.1 ml by virus serum neutralization test.

In this way, the seed venom to be used as a vaccine was passaged in isolation, and the seed venom was inoculated, cultured and taken in the same manner as above, and lyophilized or stored at -80 ° C. The bulk virus to be used for the vaccine was inoculated, cultured and taken in the same manner as above, and stored frozen at -40 ° C.

According to the method described above, the virus culture solution was collected and stored, and centrifuged at 3000 rpm for 30 minutes to remove the cell pieces, and the virus culture supernatant was taken.

(2) inactivation of viruses

Each virus supernatant isolated was treated with 0.5% formalin solution and kept at room temperature for 12 hours to inactivate each virus.

(3) Preparation of Mixed Vaccine

Inactivated CPV-2a and CPV-2b, CRV and CCoV were mixed in a ratio of 1: 1: 1, and RehydraGel was added to 10%. Or 50% of IMS1312N (Seppic, France) was added, followed by stirring for at least 60 minutes to prepare a mixed vaccine. The prepared mixed vaccine was used in the following experimental examples, while stored in a dark place at 2-5 ° C.

Formulation example Mixed vaccines (15 PCR01 ), (15 PCR02 ), (16 PCR01 ) Produce

The inactivated virus was mixed as appropriate in Tables 1 to 3 to prepare a mixed vaccine.

TABLE 1

Lot number 1 (15PCR01) Antigens and Additives Bulk quantity Reference





CPV-2a 2,400 ml 300 ml CPV-2b 0 ml 0 ml CCoV 2,400 ml 300 ml CRV 2,400 ml 300 ml IMS1312N 0 ml 0 ml Rehydrogel 800 ml 100 ml all 8,000 ml 1,000 ml

TABLE 2

Lot number 2 (15PCR02) Antigens and Additives Bulk quantity Reference





CPV-2a 500 ml 100 ml CPV-2b 500 ml 100 ml CCoV 750 ml 150 ml CRV 750 ml 150 ml IMS1312N 2,500 ml 500 ml Rehydrogel 0 ml 0 ml all 5,000 ml 1,000 ml

TABLE 3

Lot number 3 (16PCR01) Antigens and Additives Bulk quantity Reference





CPV-2a 500 ml 100 ml CPV-2b 0 ml 0 ml CCoV 500 ml 100 ml CRV 500 ml 100 ml IMS1312N 1500 ml 300 ml Rehydrogel 0 ml 0 ml all 3,000 ml 600 ml

Experimental Example  1: Confirmation of immunogenicity in experimental animals of mixed vaccines

As a test animal for immunogenicity, a guinea pig having a weight of 350-400 grams was used. The experimental group was composed of 5 animals and a control group of 3 animals.

1 ml of the mixed vaccine prepared in the above example was first inoculated into the hindlimb muscle of the experimental group, and after 2 weeks, 1 ml of the mixed vaccine was inoculated twice. Two weeks after the first and second inoculations, a total of 2 ml of blood was collected and antibody titers were measured. Antibody titers to CPV were measured using hemagglutination inhibition test, and antibody titers to CCoV and CRV were measured using serum neutralization test. The results are shown in FIGS. 4 to 6.

Vaccines containing only each viral antigen were inoculated into another experimental group in the same amount as the mixed vaccine, and antibody titers were measured in the same manner as above. The results are shown in FIGS. 4 to 6. The control group was inoculated with sterile PBS only in the same content as the mixed vaccine, and antibody titer was measured by the same method as above. The results are shown in FIGS. 4 to 6.

As shown in FIG. 4, in the case of CPV, antibody titers were lowered two weeks after the first inoculation compared to the single inoculation group when the vaccine was inoculated, but after two weeks, the antibody levels were similar. Was induced.

5 and 6, in the case of CRV and CCoV, the antibody titer was induced at a level similar to that of the single inoculation group two weeks after the second inoculation when the mixed vaccine was inoculated.

Experimental Example  2: Confirmation of the Induction Capability of Antibodies in a Mixed Vaccine

Serving puppies were used as experimental animals to confirm the ability of the antibody to induce.

During the breeding of dogs, 2 ml of blood were collected at two week intervals, and the antibody was tested to confirm the time point at which the antibody was lost for each virus. Antibody titers to CPV were measured using hemagglutination inhibition test, and antibody titers to CCoV and CRV were measured using serum neutralization test.

After raising to the point where the antibody titer that does not interfere with the parental transfer antibody based on CPV falls below 1:80, the mixed vaccine prepared in Example was inoculated. Three ml of the mixed vaccine was first inoculated into the hind paw muscles, and two weeks later, three ml of the mixed vaccine was inoculated. Blood collection was performed before inoculation, two weeks after the first inoculation, and two weeks after the second inoculation. Antibody titers to CPV were measured using hemagglutination inhibition test, and antibody titers to CCoV and CRV were measured using serum neutralization test. The results are shown in FIGS. 7 to 9.

The control group was inoculated with sterile PBS only in the same content as the mixed vaccine, and antibody titer was measured by the same method as above. The results are shown in FIGS. 7 to 9.

As shown in Figures 7 to 9, dogs vaccinated with the mixed vaccine of the present invention had an increased antibody titer for each virus. Therefore, the mixed vaccine of the present invention can be seen that the antibody is excellent in induction ability.

Experimental Example  3: Confirm safety of the combination vaccine

The safety of the vaccine was investigated using SPF beagle dogs as experimental animals.

The vaccine used for safety confirmation was that the mixed vaccine prepared in the above example was added to NA, NB, Thio medium, and cultured for one week, after which bacterial growth was determined to be negative.

Three vaccines, 15PCR01, 15PCR02 and 16PCR01, were prepared and immediately inoculated into the hind leg muscles of SPF beagle dogs. After inoculation for 21 days, clinical symptoms such as immediate inoculation response, delayed inoculation response, fever, respiratory symptoms, diarrhea, and the like were confirmed. In addition, after 3 months, 6 months, 9 months, 12 months, and 15 months after the combination vaccine was prepared, the same amount was inoculated into the hind leg muscle of SPF beagle dogs, and safety was confirmed by the same method. The results are shown in Tables 4-6.

Table 4 Safety Investigation of 15PCR01 Vaccine-1

Elapsed time after vaccine manufacture 0 months 3 months 6 months 9 months 12 months 15 months Clinical symptoms clear clear clear clear clear clear Mortality Not dead Not dead Not dead Not dead Not dead Not dead

Table 5 Safety Study of 15PCR02 Vaccine-2

Elapsed time after vaccine manufacture 0 months 3 months 6 months 9 months 12 months 15 months Clinical symptoms clear clear clear clear clear clear Mortality Not dead Not dead Not dead Not dead Not dead Not dead

Table 6 Safety Study of 16PCR01 Vaccine-3

Elapsed time after vaccine manufacture 0 months 3 months 6 months 9 months Clinical symptoms clear clear clear clear Mortality Not dead Not dead Not dead Not dead

As shown in Table 4 to Table 6, the mixed vaccine of the present invention did not show significant clinical symptoms at the time of inoculation, nor did the experimental animals die. Thus, the vaccine of the present invention has proven to be a safe vaccine with no side effects.

Experimental Example  4: efficacy test of combination vaccine

The prepared vaccine was first inoculated and then 2 weeks later. Before inoculation, two weeks after the first inoculation, and two weeks after the second inoculation, antibody titers for each virus were measured. The control group was inoculated with sterile PBS in the same amount as the mixed vaccine instead of the mixed vaccine, and antibody titers were measured in the same manner. The results are shown in Tables 7 to 9.

<Table 7>

Farm Name
Test group
Sample Number
Before vaccination 2 weeks after the first dose 2 weeks after the 2nd inoculation CPV CCoV CRV CPV CCoV CRV CPV CCoV CRV Seogyowon Farm














Vaccine group



One 4 2 0 3 One One 5 4 3 2 2 3 0 3 2 2 5 5 4 3 3 0 0 3 One One 6 4 4 4 One 0 0 0 0 2 4 2 4 5 3 2 0 2 2 2 4 5 3 6 2 0 0 4 2 3 4 4 4 7 0 0 0 2 2 2 6 3 4 8 3 0 0 2 One 3 4 5 5 9 4 2 0 4 One 2 5 6 4 10 5 0 0 4 3 2 5 5 4 11 3 0 0 4 2 3 6 4 5 12 3 0 0 4 2 3 5 6 4 13 3 2 0 3 One 3 4 5 4 Average 2.8 0.8 0.0 2.9 1.5 2.2 4.8 4.5 4.0 Control

One 0 0 0 0 0 0 0 0 0 2 3 2 0 3 2 0 3 2 0 3 4 3 0 4 2 0 5 3 0 4 5 0 0 6 0 0 5 0 0 Average 3.0 1.3 0.0 3.3 1.0 0.0 3.3 1.3 0.0

<Table 8>

Farm Name Test group
Sample Number
Before vaccination 2 weeks after the first dose 2 weeks after the 2nd inoculation CPV CCoV CRV CPV CCoV CRV CPV CCoV CRV Longevity Farm























Vaccine group











One 0 0 0 2 One 2 5 5 5 2 0 0 0 3 2 2 6 5 4 3 2 0 0 2 One 3 5 4 5 4 0 0 0 0 0 2 3 3 5 5 7 0 0 5 2 2 6 4 5 6 7 0 0 7 2 3 5 5 4 7 0 0 0 2 2 3 6 5 5 8 0 0 0 One One 3 5 6 5 9 0 0 0 2 One One 5 6 6 10 0 0 0 0 One 0 5 4 4 11 0 0 0 2 0 2 6 4 4 12 0 0 0 One 2 2 6 3 4 13 0 0 0 2 2 2 5 5 5 14 0 0 0 3 2 3 4 5 5 15 2 0 0 2 3 2 5 5 4 16 3 0 0 2 2 3 6 3 6 17 2 0 0 One 2 4 5 6 4 18 2 0 0 4 One 3 7 5 5 Average 1.4 0.0 0.0 2.3 1.5 2.3 5.3 4.6 4.7 Control




One 0 0 0 0 0 0 0 0 0 2 3 0 0 2 0 0 3 0 0 3 4 0 0 6 0 0 5 0 0 4 7 0 0 7 0 0 5 0 0 5 0 0 0 0 0 0 0 0 0 Average 2.8 0.0 0.0 3.0 0.0 0.0 2.6 0.0 0.0

<Table 9>

Farm Name Test group Sample Number Before vaccination 2 weeks after the first dose 2 weeks after the 2nd inoculation CPV CCoV CRV CPV CCoV CRV CPV CCoV CRV Jung Yoon Seok Farm

















Vaccine group











One 0 0 0 2 One One 4 4 4 2 0 0 0 One 2 One 5 4 4 3 7 0 0 7 One One 6 4 3 4 7 0 0 6 0 2 5 3 4 5 7 0 0 6 2 2 6 4 3 6 7 0 0 5 2 2 6 4 3 7 0 0 0 2 2 2 5 3 4 8 0 0 0 2 One 2 4 4 4 9 0 0 0 One One 2 4 4 5 10 0 0 0 2 3 2 4 5 4 11 0 0 0 2 2 3 5 4 4 12 0 0 0 One 2 2 4 4 3 Average 2.3 0.0 0.0 3.1 1.6 1.8 4.8 3.9 3.8 Control




One 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 3 0 6 3 0 0 0 0 0 0 5 0 0 4 7 0 0 7 0 0 5 0 0 5 7 0 0 6 0 0 0 0 Average 2.8 0.0 0.0 2.6 0.0 0.0 3.3 0.0 0.0

As shown in Tables 7 to 9 above, when inoculated with the mixed vaccine of the present invention, antibody titers against CPV, CCoV, and CRV were increased in comparison with the control group. In addition, there was no incidence of viral diarrhea in the farm during the test period, and no significant difference in clinical symptoms occurred in the inoculated group compared to the control group.

Figure 1 shows normal CRFK cells (a) and CRFK cells degenerated after inoculation with CPV-2b (b).

Figure 2 shows normal CRFK cells (a) and CRFK cells degenerated after CCoV inoculation (b).

Figure 3 shows normal MA104 cells (a) and cell degeneration MA104 cells (b) after CRV inoculation.

Figure 4 shows the antibody value against CPV of the mixed vaccination group, CPV alone inoculation group and the control group of the present invention.

Figure 5 shows the antibody titers to CCoV of the mixed vaccination group, CCoV alone inoculation group and the control group of the present invention.

Figure 6 shows the antibody titers to CRV of the mixed vaccination group, CRV alone inoculation group and the control group of the present invention.

Figure 7 shows the antibody titer for CPV of the mixed vaccination group and control of the present invention.

Figure 8 shows the antibody titer for CCoV of the mixed vaccination group and control of the present invention.

Figure 9 shows the antibody titer for CRV of the mixed vaccination group and control of the present invention.

Claims (7)

A vaccine for the prevention of canine viral diarrhea containing inactivated canine rotavirus (KCTC 11081BP). The vaccine of claim 1, wherein the vaccine is inactivated canine parvovirus CPV-2a (KCTC 11082BP), inactivated canine parvovirus CPV-2b (KCTC 11083BP), inactivated canine rotavirus (KCTC 11081BP) and inactivated A vaccine comprising all of the canine coronavirus (KCTC 11317BP). The vaccine of claim 1 or 2, further comprising an immune enhancing agent. The canine parvoviruses CPV-2a and CPV-2b having a virus titer of 2 ¹⁴ HAU by the hemagglutination inhibition test, and the dog rotavirus having a antibody titer of 10 ^6.5 TCID ₅₀ /0.1 ml by the serum neutralization test. , And a dog coronavirus having an antibody value of 10 ^6.5 TCID ₅₀ /0.1 ml by serum neutralization test is contained in a volume ratio of 40:25:25 or 30:30:30. The vaccine according to claim 4, wherein the canine parvoviruses CPV-2a and CPV-2b having a viral titer of 2 ¹⁴ HAU by the hemagglutination inhibition test are contained in a volume ratio of 1: 1. The canine parvovirus CPV-2a, CPV-2b and dog coronavirus passaged in CRFK (Crandell Feline Kidney) cells, and the dog rotavirus passaged in MA104 cells (monkey kidney cells). The vaccine characterized in that the inactivation. The vaccine according to claim 1, wherein the vaccine inactivates canine rotavirus passaged in MA104 cells (monkey kidney cells).

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