CN1942204A - Inactivated mixed vaccine for porcine respiratory disease and the method of manufacturing thereof - Google Patents

Abstract

The present invention relates to inactivated mixed vaccine for preventing porcine respiratory disease. The inactivated mixed vaccine of the present invention, which comprises a certain amount of inactivated Haemophilus parasuis S4 and S5, Mycoplasma hyopneumoniae , and PRRS virus, can effectively prevent porcine respiratory disease such as Glasser's disease, Swine enzootic pneumonia and PRRS. Further, the mixed vaccine of the present invention can prevent progress of the porcine respiratory disease to PRDC (Porcine Respiratory Disease Complex) and PMWS (Post-weaning Multisystemic Wasting Syndrome).

Description

Inactivated mixed vaccine for porcine respiratory disease and preparation method thereof

technical field

The present invention relates to vaccines for porcine respiratory diseases. In particular, the invention relates to inactivated mixed (combined) vaccines for porcine respiratory diseases.

Background technique

Swine respiratory disease has become a significant problem causing severe economic losses on farms. In particular, if the disease progresses to post-weaning multisystemic wasting syndrome (PMWS) characterized by neonatal death, persistent shrinkage and daily weight loss, or if the disease progresses to porcine respiratory syndrome ( porcine respiratory disease complex, PRDC), it produces extremely serious problems. In order to avoid and reduce the economic loss caused by the porcine respiratory disease (syndrome) infection, various vaccines have been used. However, because porcine respiratory disease has a variety of causes, vaccines for any one condition are not effective in many cases. At this point, several vaccine mixtures were developed and applied on several farms. However, the combination vaccine does not have a sufficiently satisfactory protective effect compared to the monovalent antigen vaccine because the diseases occur at different times.

Porcine enzootic pneumonia caused by Mycoplasma hyopneumoniae, Glasser's disease caused by Haemophilus parasuis, and porcine reproductive and respiratory syndrome (PRRS) Porcine Reproductive and Respiratory Syndrome (PRRS) caused by a virus is known as a prominent porcine respiratory disease (syndrome).

Porcine Glaser's disease is highly contagious for piglets of 2-4 months of age, and generally develops from weaned piglets of 5-6 weeks of age. The disease occurs through infection with pathogenic microorganisms, generally in the upper respiratory tract, especially when the piglet's immune system is suppressed by a stress response, eg, transportation or a sudden environmental change. Mycoplasma hyopneumoniae or PRRS virus can cause Glasser's disease in pigs. Porcine enzootic pneumonia is a year-round disease with a very high infection rate but exhibits low mortality. The disease occurs primarily in piglets of 4-5 weeks of age when reduced levels of sow-derived antibodies fail to confer protection against infection. The disease destroys the mucosal villi and cells of the airways and weakens the primary immune system, causing a dry cough and chronic pneumonia. In turn, the weakened immune response of lung macrophages predisposes porcine enzootic-infected pigs to other respiratory diseases (Glasser's disease, porcine pleuropneumonia, PRRS, and swine influenza, etc.), causing high mortality in pigs. The PRRS virus is an enveloped RNA virus belonging to the genus Arterivirus of the family Togaviridae. The PRRS virus is characterized by causing multiple reproductive failures in adult pigs, as well as low feeding efficiency, high post-weaning mortality and respiratory syndrome associated with interstitial pneumonia in piglets. Furthermore, in contrast to acute infection, which exhibits typical symptoms, chronic infection itself, persistent infection and secondary infection concurrent with other pathogens can lead to more serious and longer-lasting economic losses, even if the chronic condition itself is not severe.

In particular, the clinical occurrence of PRRS or porcine enzootic pneumonia makes pigs very susceptible to secondary infection with other respiratory diseases, for example, Glasser's disease, porcine enzootic pneumonia, swine influenza, Streptococcus spp and Salmonella cholerasuis. If porcine respiratory disease progresses to PMWS and PRDC, it results in daily weight loss in piglets and growing pigs, low feeding efficiency, high post-weaning mortality and more respiratory syndrome. Therefore, there is a need to develop methods of preventing said prominent porcine respiratory diseases that can lead to severe economic losses on farms. Also, the need for protection against PMWS and PRDC has increased in practice. However, no suitable vaccine fully meets these needs.

In this regard, our inventors have found through research a method of simultaneously preventing said prominent porcine respiratory disease with an effective regimen. We accomplished the present invention by designing a mixed vaccine containing three types of antigens, and by measuring safety and antibody formation in animal models.

Contents of the invention

The present invention provides a mixed (combined) vaccine for the three types of porcine respiratory diseases. More specifically, the present invention provides an inactivated mixed vaccine for preventing porcine Glaser's disease, porcine enzootic pneumonia and porcine reproductive and respiratory syndrome (porcine reproductive and respiratory syndrome, PRRS).

Another object of the present invention is to provide an inactivated mixed vaccine for reducing the causes of three types of porcine respiratory disease, PMWS and PRDC in adult pigs.

Another object of the present invention is to provide a new method for detecting the PRRS neutralizing antibody titer using guinea pigs when preparing the mixed vaccine of the present invention.

In order to achieve the above-mentioned purpose, the inventor has designed and can prevent or reduce porcine Glaser's disease, porcine enzootic pneumonia and PRRS, post-weaning multisystemic wasting syndrome (Post-weaningMultisystemic Wasting Syndrome, PMWS) and porcine respiratory syndrome (Porcine Respiratory Disease Complex, PRDC) an effective inactivated mixed vaccine with excellent immunity for the etiology of the onset and development of three types of porcine respiratory diseases. In the present invention, the Haemophilus parasuis (Haemophilus parasuis) S4 bacterial strain and the Haemophilus parasuis (Haemophilus parasuis) S5 bacterial strain isolated from sick piglets in a domestic pig farm are inactivated with formalin to prepare Vaccines for Glaser's disease. A vaccine for porcine enzootic pneumonia was prepared by inactivating Mycoplasma hyopneumoniae J/101 strain with formalin. Use MN-HS (MinnesotaUniversity, Prof. Han Soo Joo, American Type Culture Collection (American TypeCulture Collection, ATCC) accession number: VR2509) or CNV-1 from porcine lung macrophages (obtained from Prof. Kim, Hyeon Su, Chungnam National University, Korea; Sequence analysis of ORF4 gene of porcine reproductive and respiratory syndrome virus (PRRSV) Korean isolate CNV-1, Korean J.Vet Res(1999)39(2):294-300), porcine testis cell line (swine testis cell line, STL) or A-72 cells (ATCC No.: CRL-1542) to prepare vaccines for PRRS. After culturing the infected cells, they were isolated when approximately 80% cytopathic effect (CPE) was observed and used for vaccine preparation.

At the same time, when the inactivated PRRS vaccine is injected into antibody-negative pigs, the typical serum neutralization (SerumNeutralization, SN) test cannot provide antibody titers. In this regard, the inventors of the present invention devised a new antibody titer measurement system. That is, the inventors first determined the comparative relationship between guinea pigs and pigs regarding vaccination with inactivated PRRS. Then, the present invention measured neutralizing antibody titers using guinea pigs. Thus, the new method of measuring PRRS neutralizing antibody titers using guinea pigs solves a problem that cannot be overcome using pigs. Because it is very difficult to obtain pigs that remain negative for PRRS virus (90.4% positive on farms and 45.1% positive for pigs), and because antibodies (IgG) that neutralize PRRS virus appear at a later stage (4-5 weeks post-vaccination), no Easy detection of neutralizing antibody titers. In order to solve the problem, the inventors used guinea pigs instead of pigs for the above-mentioned detection of neutralizing antibody titers. The neutralization titer against PRRS virus was determined after injecting the mixed vaccine of the present invention to guinea pigs and piglets. Neutralization with complement (containing 1%-5% by weight of guinea pig fresh serum) at 4°C for 48 hours proved to be the most effective method.

The mixed vaccine composition of the present invention preferably contains vaccine components in the range of 20-30% (vol/vol). The components and their preferred contents are as follows: Haemophilus parasuis S4 strain (optical density (optical density, OD) 0.4-0.5, 410 nanometers), Haemophilus parasuis S5 strain (OD 0.4-0.5 , 410 nm), Mycoplasma hyopneumoniae J/101 (OD 0.05-0.15, 410 nm) and PRRS MN-HS (10 ^5.0 -10 ⁸ median tissue culture infectious dose (TCID ₅₀ )/ml). After the vaccine components were completely mixed, the mixture was filtered using a copper mesh, and aluminum hydroxide gel 10-20% (v/v) was added to the filtered mixture to allow the vaccine components to adsorb onto the gel. Next, oil adjuvant (IMS 1313NPR) 5-10% (volume/volume) and ethylenediaminetetraacetic acid (EDTA) 0.14% (weight/volume) were added to the mixture to obtain a mixed vaccine composition.

In one embodiment, mice, guinea pigs and piglets are used to evaluate the safety of the mixed vaccines of the present invention. As a result, mice and guinea pigs survived 7 days after inoculation, and piglets survived 14 days after inoculation.

In another embodiment, the immunogenicity of the vaccine mixture of the invention was determined. As shown in Table 9, the mixed vaccines induced antibody formation.

In another embodiment, the determination of neutralizing antibody titers against PRRS virus using guinea pigs is compared to using antibody negative pigs. According to the results, determination of antibody titers against PRRS virus using guinea pigs provides more accurate antibody titers.

In another embodiment, the activity of the vaccine mixture of the invention is monitored every 3 months during storage in the refrigerator for 21 months. The results demonstrate that the vaccine of the present invention is indeed effective and has no problems during long-term storage (Table 15, Table 16, Table 17 and Table 18).

In another embodiment, safety and immunogenicity are demonstrated on farm piglets. The vaccines of the present invention showed excellent safety and immunogenicity (Table 19 and Table 20).

In another embodiment, a group of pigs given the mixed vaccine of the present invention and a group of pigs given the conventional vaccine on three farms were evaluated for completion stage, daily body weight gain and sales rate of market pigs (Table 21, Table 22, Table 23 and Table 24). For 11-week-old pigs, the average body weight range of the pigs administered the mixed vaccine of the present invention was 3.1-4.2 kg, which proves that the weight gain rate of the pigs administered the mixed vaccine of the present invention is higher than that of pigs treated with the conventional vaccine. The sales rate of pigs treated with the mixed vaccine of the present invention was 10% higher than that of pigs treated with conventional vaccines. Therefore, the average sales time of the group treated with the mixed vaccine of the present invention was shortened by 10 days compared with that of the group treated with the conventional vaccine.

Description of drawings

Figures 1 to 3 show examples of pneumonia scores;

Figure 1 is an example of pneumonia and shows the percentage of each lobe of the whole normal lung;

Figure 2 is an example of pneumonia and shows lung damage with a 12% lobar score;

Figure 3 is an example of pneumonia and shows lung lesions with a 20% lobe score.

The present invention will be disclosed in further detail based on the examples. It is understood that the scope of the present invention is not limited by

EXAMPLE LIMITATIONS.

Example

Example 1: Isolation and cultivation of viruses

For porcine Glasser's disease, in tryptic soy broth containing 45 μg/ml β-Nicotinamide adenine dinucleotide (β-NAD), 0.5% yeast extract, and 0.5% sucrose The S4 strain of Haemophilus parasuis and the S5 strain of Haemophilus parasuis (obtained from National Veterinary Research and Quarantine Service) were respectively cultured in (Tryptic Soy Broth, TSB) medium. The cultured strains were then isolated, lyophilized and cryopreserved at -80°C. The Mycoplasma hyopneumoniae J/101 strain (obtained from the National Veterinary Research and Quarantine Service), which causes porcine enzootic pneumonia, was released and cultured in mycoplasma medium (Table 1). After isolation, the cultured Mycoplasma strains were lyophilized and cryopreserved at -80°C. Next, the Mycoplasma hyopneumoniae J/101 strain was transferred to 1/3 volume of Mycoplasma medium (Table 1), and cultured for 7 to 10 days until the color of the medium turned yellow. Inoculate and culture PRRS in porcine lung macrophages, swine testis cell line (STL) or A-72 cells (ATCC No.: CRL-1542) (Table 2, cell culture medium) (reproductive respiratory syndrome) MN-HS virus (Minnesota University, Prof. Han Soo Joo, ATCC No.: VR2509) or the pathogen of CNV-1 (obtained from Prof. Kim, Hyeon Su, Chungnam National University, South Korea) until 80 %CPE, and then isolate the cultured virus strains. The isolated strains were then lyophilized and cryopreserved at -80°C for use as vaccine components. Next, lung macrophages, STL cells or A-72 cells were suspended in the cell culture medium (Table 2). The cell suspension was incubated at 37°C for 1-3 days, and then the suspension was dispensed into culture flasks. When the cultured cells form a layer, the medium is removed. Then, 10 ^3.0 TCID ₅₀ /ml (50% tissue culture infectious dose) of the isolated MN-HS virus was transferred to virus medium (Table 3) and cultured at 37°C using a rotary cell culture system.

Table 1 Mycoplasma medium composition Element content Note Hank's balanced salt solution (BSS) (10X) 30ml brain heart infusion 5.0 grams whey protein hydrolyzate 1.25 grams PPLO 7.5 grams yeast extract 0.5 grams Phenol red (0.1%) 15ml distilled water 830ml pH 7.8, 121°C, 20 minutes autoclaving Glucose (50%) 2.5ml pig serum 100ml horse serum 100ml 25% baker's yeast extract 45ml Thallium acetate (2%) 5.5ml

Table 2 Cell culture medium composition (total volume: 1000 milliliters) Element content Note α-Modified Eagle's Medium (α-MEM) 930ml fetal bovine serum 50ml 7.5% _NaHCO3 20ml Amphotericin B (Fungizone) 0.025 μg/ml Penicillin G-Na 100 units/ml streptomycin 100 μg/ml

Table 3 virus medium composition (total volume: 1000 milliliters) Element content Note αMEM 950ml fetal bovine serum 30ml Diethylaminoethyl dextran (DEAE-dextran) 20 μg/ml Trehalose 10mg/ml 7.5% _NaHCO3 20ml Amphotericin B 0.025 μg/ml Penicillin G-Na 100 units/ml streptomycin 100 μg/ml

Example 2: Isolation and inactivation

For porcine Glasser's disease, each seed culture solution of Example 1 was released in TSB medium containing 45 μg/ml β-nicotinamide adenine dinucleotide (β-NAD), 0.5% yeast extract and Incubate at 37°C for 12 hours. The cultured strains were then isolated and concentrated, and inactivated using 0.3% formalin for 72 hours. For porcine enzootic pneumonia, M. hyopneumoniae J/101 strain was transmitted in mycoplasma medium and cultured at 37°C for 7-10 days. Then, the cultured Mycoplasma strains were isolated and concentrated, and inactivated using 0.2% formalin at room temperature for 72 hours. For PRRS virus, porcine lung macrophages, porcine testis cell line (STL) cells or A-72 cells were inoculated with MN-HS or CNV-1 and cultured at 37°C for 3-5 days using a rotary culture system. When approximately 80% CPE was observed after virus inoculation, virus was isolated and inactivated using 0.1% formalin for 48 hours at room temperature. For Glasser's disease and porcine enzootic pneumonia, the inactivated strain was diluted appropriately. Then, use a UV spectrophotometer to measure the optical density (optical density, OD) value (410 nm) of the diluted strain. The inactivated PRRS virus was diluted 1/10, and 0.1 ml of the diluted solution was inoculated to porcine lung macrophages, porcine testis cell line (STL) cells or A-72 cells, followed by culturing the cells at 37°C for 72 hours. Virus levels were measured following the appearance of CPE after PRRS virus inoculation. The virus content exceeds 10 ^6.0 TCID ₅₀ /ml.

Embodiment 3: the preparation of inactivated mixed vaccine

The three antibodies of Example 2 were used to isolate strains, isolate pathogenic substances and inactivate various infection culture fluids. The total content of vaccine components is 30% (vol/vol) of the total mixed vaccine composition. The OD values of each vaccine component are as follows: Haemophilus parasuis S4 (410 nm, OD 0.45), Haemophilus parasuis S5 (410 nm, OD 0.45), Mycoplasma hyopneumoniae J/101 (410 nm, OD 0.1) and PRRS MN-HS (10 ^6.0 TCID ₅₀ /ml). After mixing the vaccine composition, filter through a copper mesh. Then, use aluminum hydroxide gel 20% (vol/vol) to adsorb the filtered composition, then additionally add oil adjuvant (IMS 1313NPR) 5% (vol/vol) and EDTA 0.14% (weight/volume), and Mixing was carried out at 1000 rpm for 30 minutes to obtain a mixture. Next, an amount of the mixture was dispensed into bottles and stored in a cold room at 2-7°C. The mixed vaccine composition obtained formed two separate layers, a rose pink liquid layer and a precipitate layer, wherein the two layers easily mixed into a homogeneous solution when shaken. The composition does not have any taste and smell. Each batch of vaccine samples was transferred to thioglycollate medium (Thioglycollate medium, Thio), nutrient agar (Nutrient agar, NA) and nutrient broth (Nutrient broth, NB) respectively, and cultured at 22°C for 7 days. Additionally, each batch of vaccine samples was transferred to thioglycolic acid medium (Thio), nutrient agar (NA) and nutrient broth (NB), respectively, and incubated at 37°C for 7 days. During the cultivation, no contamination of the medium occurred (Table 4). Each vaccine sample was used in the following experiments. The formalin content in the batches ranged from 0.19-0.20% (v/v) (Table 5).

Table 4 Contamination of various microorganisms Vaccine sample (lot number) 22°C 37°C Note Thio NA NB Thio NA NB 1 - ^* - - - - - 2 - - - - - - 3 - - - - - -

^* -: no pollution

Table 5 Formalin content Reagent experiment method Vaccine sample (lot number) result 1. Formalin standard solution 2. Schiff's reagent Add Schiff's reagent (2 mL) to each sample Add Schiff's reagent (2 mL) to the standard formalin solution Measure the optical density at 520 nm using a spectrophotometer 1 0.20% 2 0.20% 3 0.19%

Example 4: Evaluation of Safety in Animal Models

(1) Safety test in mice

For this test, 10 mice weighing 13 g and 10 mice weighing 15 g were used. Five mice weighing 13 g and five mice weighing 15 g were intraperitoneally administered various vaccine samples of Example 3 (0.5 ml, Lot No. 1-3). Five mice weighing 13 g and five mice weighing 15 g were subcutaneously administered various vaccine samples of Example 3 (0.5 ml, Lot No. 1-3). Inoculated mice were monitored and compared to 5 untreated control mice. All mice injected with the mixed vaccine of the present invention survived the monitoring period (Table 6).

Table 6 uses mice to the safety test of mixed vaccine Vaccine sample (lot number) number of mice Administration route and amount Monitoring days result 1 5 intraperitoneal, 0.5 mL 7 survive 5 Subcutaneous, 0.5 mL 7 survive 2 5 intraperitoneal, 0.5 mL 7 survive 5 Subcutaneous, 0.5 mL 7 survive 3 5 intraperitoneal, 0.5 mL 7 survive 5 Subcutaneous, 0.5 mL 7 survive control 5 unprocessed 7 survive

(2) Safety test in guinea pigs

Twelve guinea pigs each weighing 300 g and 350 g were used in this test. Each of the vaccine samples (0.1 ml) of Example 3 was injected intramuscularly to 4 guinea pigs each weighing 300 g and 350 g. Each of the vaccine samples (0.1 ml) of Example 3 was injected intradermally to 4 guinea pigs each weighing 300 g and 350 g. Each of the vaccine samples (0.1 ml) of Example 3 was subcutaneously injected into 4 guinea pigs each weighing 300 g and 350 g. The vaccinated guinea pigs were monitored and compared to 4 untreated control guinea pigs. All guinea pigs survived the monitoring period (Table 7).

Table 7 Safety tests using guinea pigs Vaccine sample (lot number) Injection (number of times) Administration route and amount Monitoring days result 1 4 1.0 mL intramuscularly 7 survive 4 1.0 mL subcutaneously 7 survive 4 Intradermal injection of 0.1ml 7 survive 2 4 1.0 mL intramuscularly 7 survive 4 1.0 mL subcutaneously 7 survive 4 Intradermal injection of 0.1ml 7 survive 3 4 1.0 mL intramuscularly 7 survive 4 1.0 mL subcutaneously 7 survive 4 Intradermal injection of 0.1ml 7 survive control 4 unprocessed 7 survive

(3) Test of safety in piglets

Eighteen one-week-old piglets negative for antibodies to H. parasuis, M. hyopneumoniae and PRRS virus were used in this experiment. Five piglets were injected through the neck muscle with the mixed vaccine of Example 3 (2.0 ml, Lot No. 1). Likewise, 5 piglets were injected through the neck muscle with the mixed vaccine of Example 3 (2.0 ml, Lot No. 2). In addition, 5 piglets were injected through the neck muscle with the mixed vaccine of Example 3 (2.0 ml, Lot No. 3). Piglets injected with the mixed vaccine of the present invention and 3 untreated piglets used as controls were monitored for 14 days. As a result, none of the piglets had clinical symptoms such as anorexia, respiratory symptoms (such as cough and dyspnea), diarrhea, vomiting, and neurological symptoms (see Table 8). They maintain a normal body temperature in the range of 38.7-39.7°C.

Table 8 uses the safety test of piglets Vaccine sample (Lot No.) age, route of administration, amount number of days serial number clinical monitoring body temperature (°C) anorexia respiratory syndrome Diarrhea Vomit neurological symptoms appearance 1 1 week old neck muscle 2.0ml 14 1 38.7-39.4 - ^* - - - - - 2 38.8-39.2 - - - - - - 3 39.0-39.7 - - - - - - 4 38.9-39.2 - - - - - - 5 38.8-39.6 - - - - - - 2 1 week old neck muscle 2.0ml 14 6 38.7-39.5 - - - - - - 7 38.7-39.6 - - - - - - 8 38.9-39.7 - - - - - - 9 38.8-39.5 - - - - - - 10 38.9-39.6 - - - - - - 3 1 week old neck muscle 2.0ml 14 11 38.8-39.6 - - - - - - 12 39.1-39.7 - - - - - - 13 39.0-39.5 - - - - - - 14 38.8-39.5 - - - - - - 15 38.9-39.7 - - - - - - control 1 week old untreated 14 16 38.8-39.3 - - - - - - 17 38.7-39.5 - - - - - - 18 38.9-39.7 - - - - - -

●-: Normal

Embodiment 5: Immunogenicity test

Eighteen one-week-old piglets negative for antibodies to H. parasuis, M. hyopneumoniae and PRRS virus were used in this experiment. Five piglets were injected through the neck muscle with the mixed vaccine of Example 3 (1.0 ml, Lot No. 1). Likewise, 5 piglets were injected through the neck muscle with the mixed vaccine of Example 3 (1.0 ml, Lot No. 2). In addition, 5 piglets were injected through the neck muscle with the mixed vaccine of Example 3 (1.0 ml, Lot No. 3). Two weeks after the first injection, each piglet was injected near the ear muscle with the same vaccine (2.0 ml) as used for the first time. Four weeks after the second injection, blood was collected from vaccinated piglets and two untreated piglets for the determination of antibody titers. Two weeks after the first injection, the enzyme-linked immunosorbent assay (ELISA) antibody titer to Haemophilus parasuis increased 80-160 times, and after the second injection 4 weeks, the ELISA antibody titer increased 320-1280 times. Antibody titers did not exceed 10-fold in untreated pigs used as controls. 2 weeks after the first injection, the ELISA antibody titer to Mycoplasma hyopneumoniae was increased by 80-320 times, and after the second injection 4 weeks, the ELISA antibody titer was increased by 640-2560 times. Antibody titers did not exceed 10-fold in untreated pigs used as controls. Antibody titers against PRRS virus did not exceed 2-fold 2 weeks after the first injection, and ranged from 4-8 fold 4 weeks after the second injection. No more than 10-fold neutralizing antibody titers were measured for untreated control pigs (Table 9).

Table 9 to the immunogenicity test of piglet Vaccine sample (lot number) serial number Antibody titer Haemophilus parasuis (ELISA) Mycoplasma hyopneumoniae (ELISA) PRRS(SN) before injection 2 weeks after the first injection 4 weeks after the second injection before injection 2 weeks after the first injection 4 weeks after the second injection before injection 2 weeks after the first injection 4 weeks after the second injection 1 1 <10 80 320 <10 80 640 <2 <2 8 2 <10 160 640 <10 160 1280 <2 <2 4 3 <10 160 640 <10 160 2560 <2 <2 4 4 <10 160 1280 <10 80 1280 <2 <2 4 5 <10 160 320 <10 320 2560 <2 <2 4 2 6 <10 80 320 <10 160 1280 <2 <2 4 7 <10 80 640 <10 160 1280 <2 <2 4 8 <10 80 320 <10 80 640 <2 <2 4 9 <10 160 640 <10 320 2560 <2 <2 4 10 <10 160 640 <10 320 1280 <2 <2 4 3 11 <10 80 320 <10 80 640 <2 <2 4 12 <10 80 320 <10 160 1280 <2 <2 4 13 <10 160 1280 <10 160 2560 <2 <2 4 14 <10 160 640 <10 320 2560 <2 <2 8 15 <10 160 640 <10 160 1280 <2 <2 4 control 16 <10 <10 <10 <10 10 10 <2 <2 <2 17 <10 <10 <10 <10 10 10 <2 <2 <2 18 <10 <10 <10 <10 10 10 <2 <2 <2

Embodiment 6: Determination of guinea pig neutralizing antibody titer

Twelve guinea pigs (300-350 g) were used in this experiment. 10 of them were intramuscularly inoculated with 1 ml of the mixed vaccine of the present invention. In addition, they were injected with the same amount of the mixed vaccine three weeks after the first injection. Two guinea pigs were used as controls. Two weeks after the second injection, blood samples were collected from the guinea pigs for the determination of neutralizing antibody titers. Eight three-week-old piglets were obtained from a farm where the farm had been confirmed to be antibody negative. Six of them were injected with a vaccine sample (2 ml) through the neck muscle and additionally 3 weeks after the first injection. Two of these pigs were used as controls. Pig blood samples were collected before the first injection, 3 weeks after the first injection, 2 weeks after the second injection, and 4 weeks after the second injection. Neutralizing antibody titers were determined using the collected blood, and the results were compared with those of conventional methods as described below. That is, the conventional method involves inactivating the serum at 56° C. for 30 minutes, diluting the serum in a 96-well microtiter plate containing 50 μl of the cell culture medium (α-MEM) disclosed in Table 3 of Example 1 1/2, 50 microliters of PRRS virus culture solution (200 TCID ₅₀ /0.1 milliliter) was added to the diluted serum and neutralized at 37°C for 1 hour, and 50 microliters of 3% fetal bovine serum (fetal bovineserum, FBS) was added in addition. Microliter medium, containing 250000 PRRS virus sensitive cells (MARC-145, MA-104), in order to determine the neutralizing antibody titer, the mixture was incubated for 5 days at 37° C. in a 5% CO2 incubator and detected for CPE. Using guinea pigs and pigs, neutralizing antibody titer measurements were performed at 37°C for 1 hour and at 4°C for 48 hours, and the results were compared with each other (Table 10 and Table 11). In the case of neutralization at 4° C. for 48 hours, media with different amounts of guinea pig fresh serum (not added, 1% by weight serum, 5% by weight serum) were added as complement. Furthermore, neutralization using complement (1% fresh guinea pig serum) at 4°C for 48 hours confirmed the reproducibility of the results. Neutralizing antibody titers were also determined using complement (1% fresh guinea pig serum) neutralized at 4°C for 48 hours in antibody-negative piglets, which differs from conventional methods. Tables 10 to 14 below disclose the results of this test. From these results, it was seen that guinea pigs can be used as excellent samples for determining neutralizing antibody titers.

Table 10 Determination of neutralizing antibody titers according to neutralization time Guinea pig number and serum neutralizing antibody titer 1 2 3 4 5 6 7 8 9 10 C1 C2 A ^* <2 <2 4 <2 4 4 8 <2 4 2 <2 <2 B ^** 4 <2 8 8 16 8 8 4 16 8 <2 <2

^* 37°C, 1 hour; ^** 4°C, 48 hours

Table 11 Determination of neutralization according to the amount of complement Guinea pig number and serum neutralizing antibody titer 1 2 3 4 5 6 7 8 9 10 C1 C2 A ^* 4 <2 8 8 16 8 8 4 16 8 <2 <2 B ^** 4 <2 8 8 32 16 16 8 32 8 <2 <2 C ^*** 8 <2 4 16 64 16 <2 8 16 8 <2 <2

^* Without added complement, ^** 1% serum, ^*** 5% serum

Table 12 Complement (1% serum), 48 hours at 4°C Guinea pig number and serum neutralizing antibody titer 1 2 3 4 5 6 7 8 9 10 C1 C2 A ^* 8 <2 8 16 16 8 8 4 16 8 <2 <2 B ^** 8 <2 8 8 32 16 16 8 32 8 <2 <2 C ^*** 8 <2 4 16 16 16 8 8 16 16 <2 <2

^* Sung-sik Yoo inspection by Choongang Vaccine Laboratory Co., Ltd.

^** Yeon-sil Lee inspection by Choongang Vaccine Laboratory Co., Ltd.

^*** Hyeok-jin Kwon inspection by Choongang Vaccine Laboratory Co., Ltd.

Routine method for neutralizing antibody titer to PRRS virus in table 13 determination antibody negative pig Vaccine sample (lot number) piglet size Serum analysis (SN test) Before the injection (May 16) 3 weeks after the first injection (June 14) 2 weeks after the second injection (June 28) 4 weeks after the second injection (July 29) Vaccine Sample 01 6 <2 <2 <2 <2 <2 <2 <2 2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 2 <2 <2 <2 <2 control 2 <2 <2 <2 <2 <2 <2 <2 <2

Table 14 Determination of serum neutralizing antibody titer (1% serum) of antibody-negative pigs at 4°C for 48 hours Vaccine sample (lot number) piglet size Serum analysis (SN test) Before the injection (May 16) 3 weeks after the first injection (June 14) 2 weeks after the second injection (June 28) 4 weeks after the second injection (July 29) Vaccine Sample 01 6 <2 <2 <2 8 <2 <2 <2 4 <2 <2 <2 4 <2 <2 <2 2 <2 <2 <2 4 <2 <2 <2 8 control 2 <2 <2 <2 <2 <2 <2 <2 <2

Embodiment 7 long-term storage test

The vaccine mix of the present invention was monitored during long-term storage in the refrigerator. Vaccine samples were stored in a cool dark place in the range of 2-7°C. During the storage of the vaccine, from the day of preparation, 1.0 milliliters of the vaccine was injected through the neck muscles to healthy piglets of 1 week of age every three months, and the piglets used were negative for Haemophilus parasuis, Mycoplasma hyopneumoniae and PRRS virus, Lasts 21 months. Two weeks after the first injection, piglets were injected with 2.0 ml of the vaccine sample. Four weeks after the second injection, serum was collected and ELISA antibody titers (Haemophilus parasuis and Mycoplasma hyopneumoniae) and neutralizing antibody titers were determined. Immunogenicity was effective and stability was satisfactory until 21 months (Table 15, Table 16, Table 17 and Table 18).

Stability test (-3 months) of the mixed vaccine of table 15 Vaccine sample (lot number) products 3 months storage period Haemophilus parasuis Mycoplasma hyopneumoniae PRRS Haemophilus parasuis Mycoplasma hyopneumoniae PRRS before injection 4 months after the second injection before injection 4 months after the second injection before injection 4 months after the second injection before injection 4 months after the second injection before injection 4 months after the second injection before injection 4 months after the second injection 1 <10 320 <10 1280 <2 8 <10 320 <10 640 <2 4 <10 640 <10 2560 <2 8 <10 640 <10 1280 <2 4 <10 320 <10 2560 <2 4 <10 640 <10 2560 <2 4 2 <10 640 <10 1280 <2 8 <10 640 <10 1280 <2 8 <10 640 <10 2560 <2 4 <10 640 <10 2560 <2 4 <10 320 <10 1280 <2 4 <10 640 <10 1280 <2 4 3 <10 640 <10 2560 <2 4 <10 1280 <10 2560 <2 4 <10 640 <10 640 <2 4 <10 640 <10 1280 <2 4 <10 1280 <10 2560 <2 8 <10 640 <10 2560 <2 8

Stability test (6-9 months) of table 16 mixed vaccine Vaccine sample (lot number) After 6 months of storage After 9 months of storage Haemophilus parasuis Mycoplasma hyopneumoniae PRRS Haemophilus parasuis Mycoplasma hyopneumoniae PRRS before injection 4 months after the second injection before injection 4 months after the second injection before injection 4 months after the second injection before injection 4 months after the second injection before injection 4 months after the second injection before injection 4 months after the second injection 1 <10 640 <10 640 <2 8 <10 640 <10 640 <2 8 <10 640 <10 1280 <2 4 <10 640 <10 1280 <2 4 <10 1280 <10 2560 <2 4 <10 640 <10 2560 <2 4 2 <10 640 <10 1280 <2 8 <10 320 <10 1280 <2 4 <10 1280 <10 2560 <2 4 <10 640 <10 2560 <2 4 <10 640 <10 1280 <2 4 <10 640 <10 1280 <2 4 3 <10 640 <10 1280 <2 8 <10 640 <10 1280 <2 8 <10 640 <10 640 <2 8 <10 640 <10 640 <2 4 <10 640 <10 2560 <2 4 <10 640 <10 2560 <2 4

Stability test (12-15 months) of table 17 mixed vaccine Vaccine sample (lot number) After 12 months of storage After 15 months of storage Haemophilus parasuis Mycoplasma hyopneumoniae PRRS Haemophilus parasuis Mycoplasma hyopneumoniae PRRS before injection 4 months after the second injection before injection 4 months after the second injection before injection 4 months after the second injection before injection 4 months after the second injection before injection 4 months after the second injection before injection 4 months after the second injection 1 <10 640 <10 1280 <2 4 <10 320 <10 640 <2 4 <10 640 <10 1280 <2 8 <10 320 <10 640 <2 4 <10 320 <10 1280 <2 8 <10 640 <10 1280 <2 4 2 <10 640 <10 1280 <2 4 <10 640 <10 1280 <2 4 <10 320 <10 640 <2 8 <10 640 <10 640 <2 8 <10 640 <10 2560 <2 4 <10 320 <10 1280 <2 4 3 <10 640 <10 1280 <2 8 <10 640 <10 640 <2 4 <10 640 <10 1280 <2 8 <10 320 <10 1280 <2 4 <10 640 <10 2560 <2 4 <10 320 <10 1280 <2 4

Stability test (18-21 months) of table 18 mixed vaccine Vaccine sample (lot number) After 18 months of storage After 21 months of storage Haemophilus parasuis Mycoplasma hyopneumoniae PRRS Haemophilus parasuis Mycoplasma hyopneumoniae PRRS before injection 4 months after the second injection before injection 4 months after the second injection before injection 4 months after the second injection before injection 4 months after the second injection before injection 4 months after the second injection before injection 4 months after the second injection 1 <10 640 <2 640 <2 8 <10 320 <2 640 <2 4 <10 640 <2 1280 <2 8 <10 320 <2 320 <2 4 <10 320 <2 640 <2 4 <10 640 <2 640 <2 4 2 <10 320 <2 1280 <2 4 <10 320 <2 640 <2 4 <10 640 <2 640 <2 8 <10 320 <2 640 <2 4 <10 320 <2 640 <2 4 <10 320 <2 1280 <2 4 3 <10 640 <2 1280 <2 4 <10 640 <2 640 <2 4 <10 640 <2 640 <2 8 <10 320 <2 640 <2 4 <10 320 <2 640 <2 8 <10 320 <2 320 <2 4

Embodiment 8: Field test (Field test)

(1) Piglet safety test

In order to test the safety in piglets, 330 1-week-old healthy piglets were selected. 30 of 330 piglets were used as controls. 300 piglets were inoculated with the vaccine mixture of the invention (2.0 ml) via the neck muscle and monitored for 14 days in comparison to untreated piglets. None of the piglets had clinical signs, eg, anorexia, respiratory symptoms (eg cough and dyspnea), diarrhea, vomiting and neurological symptoms (see Table 19). The body temperature of 30 vaccinated piglets (10 piglets for each of the 3 vaccine samples) and 10 piglets used as controls were measured. All piglets exhibited normothermia (Table 19). It is proved that the mixed vaccine of the present invention is safe to the target animals.

Test of safety in the piglet of table 19 Vaccine sample (lot number) Pig age injection site injection volume Monitoring days number Clinical symptoms number anorexia respiratory symptoms Diarrhea Vomit neurological symptoms body temperature 1 1 week old neck muscle 2.0ml 14 100 No No No No No 38.7-39.6℃ 100/100 2 1 week old neck muscle 2.0ml 14 100 No No No No No 38.9-39.4℃ 100/100 3 1 week old neck muscle 2.0ml 14 100 No No No No No 38.8-39.5℃ 100/100 control unprocessed 14 30 No No No No No 38.7-39.7℃ 30/30

(2) Test of immunogenicity in piglets

Thirty-one 1-week-old piglets negative for antibodies against H. parasuis, M. hyopneumoniae and PRRS virus were used in this experiment. The 3 mixed vaccine samples (1.0 ml) were administered to 28 piglets through the neck muscle. Two weeks after the first injection, piglets were injected with 3 vaccine samples (2.0 ml) through the neck muscle, respectively. Four weeks after the second injection, serum was collected from all piglets including untreated piglets. The ELISA antibody titers to Haemophilus parasuis and Mycoplasma hyopneumoniae were determined. The neutralizing antibody titer to PRRS virus was determined. Two weeks after the first injection, the ELISA antibody titer to Haemophilus parasuis increased 80-160 times, and 4 weeks after the second injection, it increased 320-1280 times. Two weeks after the first injection, the ELISA antibody titer to Mycoplasma hyopneumoniae was increased by 80-320 times, and 4 weeks after the second injection, it was increased by 640-2560 times. Antibody titers were not more than 10-fold measured for untreated control pigs. Two weeks after the first injection, antibody titers to PRRS virus did not exceed 2-fold, and 4 weeks after the second injection, the range was 4-8 fold. No more than 2-fold neutralizing antibody titers were measured for untreated control pigs.

Table 20 to the immunogenicity test of piglet Vaccine sample (lot number) piglet number Antibody titer Haemophilus parasuis (ELISA) Mycoplasma hyopneumoniae (ELISA) PRRS(SN) before injection 2 weeks after the first injection 4 weeks after the second injection before injection 2 weeks after the first injection 4 weeks after the second injection before injection 2 weeks after the first injection 4 weeks after the second injection 1 1 <10 80 320 <10 80 640 <2 <2 4 2 10 80 640 10 80 640 <2 <2 8 3 <10 160 1280 10 160 1280 <2 <2 4 4 10 80 320 10 160 1280 <2 <2 8 5 <10 80 320 <10 80 640 <2 <2 8 6 10 160 640 10 320 2560 <2 <2 8 7 10 160 640 10 160 1280 <2 <2 4 8 10 160 320 10 80 1280 <2 <2 4 9 <10 80 320 <10 160 640 <2 <2 8 10 10 160 640 10 160 640 <2 <2 4 2 11 10 80 320 10 320 2560 <2 <2 4 12 10 160 1280 10 160 1280 <2 <2 8 13 10 160 640 10 320 2560 <2 <2 8 14 10 80 320 10 320 1280 <2 <2 8 15 10 160 640 10 160 1280 <2 <2 8 16 10 160 640 10 160 2560 <2 <2 4 17 <10 80 640 10 80 1280 <2 <2 8 18 <10 80 320 <10 80 640 <2 <2 4 3 19 10 80 320 10 160 1280 <2 <2 4 20 10 160 640 10 160 1280 <2 <2 8 twenty one <10 80 320 <10 80 640 <2 <2 4 twenty two <10 80 320 <10 80 1280 <2 <2 8 twenty three 10 80 640 20 320 2560 <2 <2 4 twenty four 10 160 640 10 160 1280 <2 <2 8 25 10 160 1280 10 320 2560 <2 <2 4 26 10 160 640 20 320 2560 <2 <2 4 27 10 80 320 10 320 1280 <2 <2 8 28 10 160 640 10 160 1280 <2 <2 8 control 29 <10 <10 10 <10 <10 10 <2 <2 <2 30 <10 <10 <10 <10 <10 <10 <2 <2 <2 31 <10 10 10 <10 10 10 <2 <2 <2

comparative example

A 6-month controlled trial using the vaccine mix of the invention was carried out on three farms in which pigs were treated with other vaccine mixes or monovalent antigen vaccines. Marketing pig age, sales rate and average body weight are monitored for this purpose.

Table 21 Farms and Vaccines farm name farmer address number of pigs vaccine Haksung HWANG, HakSu Chunbuk-myeon, Boryeong city, Chungchungnam-Do, South Korea 5000 M+PAC (porcine enzootic pneumonia 1, 3 weeks old) PRRS live vaccine (10 days old) Young Jin LEE, heon Min Hongbuk-myeon, Hongseong-Gun, Chungchungnam-Do, South Korea 6000 Respisure (porcine enzootic pneumonia 1, 3 weeks old) Eunha YUN, Byeong-jun Eunha-myeon, Hongseong-Gun, Chungchungnam-Do, South Korea 3000 RespiFend MH/HPS (porcine enzootic pneumonia + Glasser's disease 1, 3 weeks old)

Table 22 The average body weight change of the vaccine treatment group of the present invention and the traditional vaccine treatment group farm name vaccine Number of pigs tested Average body weight (kg) 3 weeks 7 weeks 11 weeks Haksung Traditional Vaccines 20 6.6 11.8 28.5 mixed vaccine 20 6.5 13.9 32.7 Young Jin Traditional Vaccines 20 6.2 10.9 30.1 mixed vaccine 20 6.3 12.5 33.2 Eunha Traditional Vaccines 20 6.4 12.5 31.8 mixed vaccine 20 6.6 14.1 34.5

Table 23 Number of Pigs and Average Pig Age at Sales farm name vaccine Number of test pigs Number of shipments (%) Average sales pig age Note Haksung Traditional Vaccines 98 82(83.6) 184 mixed vaccine 103 95(92.2) 172 8 days shorter Young Jin Traditional Vaccines 122 103(84.4) 181 mixed vaccine 119 113 (94.9) 168 shortened by 13 days Eunha Traditional Vaccines 60 53 (88.3) 179 mixed vaccine 55 51(92.7) 170 shortened by 9 days

Table 24 Lung injury scores of mixed vaccine treatment group and traditional vaccine treatment group farm name vaccine Number of test pigs lung injury score Haksung Traditional Vaccines 35 30.4 mixed vaccine 37 8.2 Young Jin Traditional Vaccines 40 33 mixed vaccine 40 5.5

As shown in Table 21, the average weight gain of 7-week-old piglets vaccinated with the mixed vaccine of the present invention ranged from 1.5-2.0 kg over those vaccinated with the conventional vaccine. The average weight gain of 11 week old piglets vaccinated with the mixed vaccine of the invention ranged from 3.1 to 4.2 kg over those vaccinated with the conventional vaccine. Referring to Table 22, the group injected with the mixed vaccine of the present invention showed a sales rate increase of 92.2-94.9%, while the group injected with the conventional vaccine increased by 83.6-84.4%. Therefore, the average marketing age of pigs was shortened by approximately 8-13 days compared to groups treated with conventional vaccines. According to Table 23, the lung injury scores of the group treated with the traditional vaccine ranged from 30.4-33%, while the scores of the group treated with the mixed vaccine of the present invention ranged from 5.5-8.2%. According to known methods (STRAW, B.E. et al., Clinical assessment of pneumonia levels in swine through measurement of the amount of coughing. In: International Pig Veterinary Society Congress, 8, 1986, Barcelona. Proceedings... Barcelona: IPVS, 1986.p.275 .) Determination of lung injury score (see Figure 1).

Industrial Applicability

Therefore, the mixed vaccine of the present invention can effectively prevent porcine Grasset's disease, porcine enzootic pneumonia and porcine reproductive and respiratory disease syndrome, these prominent porcine respiratory disease infections. In addition, the mixed vaccine of the present invention provides protection against the transition from porcine respiratory disease to porcine respiratory disease complex (Porcine Respiratory Disease Complex, PRDC) and post-weaning multisystemic wasting syndrome due to neonatal mortality, shrinkage and decreased daily weight gain. (Post-weaning Multisystemic Wasting Syndrome, PMWS) development of a practical approach.

Claims (4)

1. An inactivation for preventing porcine Glaser's disease, porcine enzootic pneumonia and porcine reproductive and respiratory syndrome, and for preventing the disease from developing into post-weaning multisystem wasting syndrome and porcine respiratory syndrome A combination vaccine that contains: Haemophilus parasuis S4 and Haemophilus parasuis S5 with an optical density of 0.4-0.5 at 410 nm (Korean National Veterinary Research and Quarantine Service) inactivated with formalin; Mycoplasma hyopneumoniae J/101 with an optical density of 0.05-0.15 at 410 nm (Korean National Veterinary Research and Quarantine Service) inactivated with formalin; The preservation number is ATCC No.: the MN-HS virus of VR2509 or the CNV-1 virus with a half tissue culture infection rate per milliliter of 10 ^5.0 -10 ^8.0 , wherein, in porcine lung macrophages, pig testis cell lines or the preservation number is ATCC No.: The MN-HS virus or CNV-1 virus was cultured in A-72 cells of CRL-1542 until 80% cytopathic effect was observed, then isolated and inactivated; and Pharmaceutically acceptable excipients. 2. An inactivation for preventing porcine Glaser's disease, porcine enzootic pneumonia and porcine reproductive and respiratory syndrome, and for preventing the disease from developing into post-weaning multisystem wasting syndrome and porcine respiratory syndrome The preparation method of mixed vaccine, this method comprises: Cultivate Haemophilus parasuis S4, Haemophilus parasuis S5 and Mycoplasma hyopneumoniae J/101, and use formalin to inactivate; Use porcine lung macrophages, porcine testis cell lines or A-72 cells to culture MN-HS virus or CNV-1 virus, isolate the cultured virus when 80% cytopathic effect occurs, and use formalin to inactivate the isolated virus Virus; Determination of ELISA antibody titers against Haemophilus parasuis S4, Haemophilus parasuis S5 and Mycoplasma hyopneumoniae J/101; Determination of neutralizing antibody titers against MN-HS virus or CNV-1 virus using guinea pig fresh serum as complement; and The inactivated vaccine components are mixed with pharmaceutically acceptable excipients. 3. The method according to claim 2, wherein the antiviral neutralizing antibody using guinea pig serum is carried out using guinea pig or piglet after neutralizing the antibody with 1-5% by weight of guinea pig fresh serum complement at 4°C for 48 hours Determination of titer. 4. The method according to claim 2 or 3, wherein said MN-HS virus or CNV-1 virus is cultured in porcine testis cell line cells or A-72 cells but not in simian cells.

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