WO2005112995A1

WO2005112995A1 - Inactivated mixed vaccine for porcine respiratory disease and the method of manufacturing thereof

Info

Publication number: WO2005112995A1
Application number: PCT/KR2005/001207
Authority: WO
Inventors: In-Joong Yoon; Sung-Min Lee; Sung-Sik Yoo
Original assignee: Choongang Vaccine Laboratory Co., Ltd.
Priority date: 2004-04-26
Filing date: 2005-04-26
Publication date: 2005-12-01
Also published as: CN1942204A

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 THE METHOD OF MANUFACTURING THEREOF

TECHNICAL FIELD The present invention relates to a vaccine for porcine respiratory diseases. Specifically, the present invention relates to an inactivated mixed (combined) vaccine for porcine respiratory diseases.

BACKGROUND ART

The porcine respiratory diseases have been serious problem which results in severe economic loss to farms. In particular, if the diseases develop to post-weaning multisystemic wasting syndrome (PMS) characterized by neonatal mortality, persistent atrophy and decrease of daily weight gain, or if the diseases develop to porcine respiratory disease complex (PRDC) , it create extremely serious problems. In order to prevent and reduce the economic losses from infections of such porcine respiratory diseases (syndromes), various vaccines have been introduced. However, since there are various causes of the porcine respiratory diseases, a vaccine for any one of conditions was not effective in many cases. In this regard, a few of mixed vaccines were developed and applied at several farms. The mixed vaccines, however, were not

satisfactory enough with protecting effects compared to vaccines with

monovalent antigen, since the diseases develop at different times.

Swine enzootic pneumonia induced by Mycoplasma hyopneumoniae,^'

Glasser's disease induced by Haemophilus parasuis', and Porcine

Reproductive & Respiratory Syndrome (PRRS) induced by PRRS virus are

known as the outstanding porcine respiratory diseases (syndromes). The Glasser's disease in a pig is highly infective to a 2 ~ 4 month

old piglet, and develops commonly from a 5 to 6 week old weaning piglet.

This disease develops by the infection of a pathogenic microbial, which

is generally located on the upper respiratory tract, especially when the

immune system of the piglet is depressed by stress, for example,

transportation or sudden environmental changes. The Glasser's disease in

a pig can be activated by Mycoplasma hyopneumoniae or PRRS virus. The

swine enzootic pneumonia, which is a year-round disease, shows very high

infection rate while representing low death rate. This disease mainly

develops from a 4 to 5 week-old piglet when the level of maternally

derived antibody degrades and do not give a protection against the

infection. This disease destroys the mucosal villi and cells of

respiratory tracts to weaken the first immune system, thereby induces dry

cough and chronic pneumonia. Subsequently, the reduced immune response

of pulmonary macrophages makes the affected pig to be easily infected with other respiratory diseases (Glasser's disease, porcine

pleuropneumonia, PRRS, and swine influenza, etc) that induces high death

rate of pigs. The PRRS virus is a RNA virus which has envelope and

belongs to genus Arterivirus of family Togaviridae. It is characterized

by causing a variety of reproductive failures in adult pigs and by

causing low feed efficiency, high post-weaning death and respiratory

syndrome relevant to interstitial pneumonia in a piglet. Further, in

contrast to acute infection showing typical symptoms, the chronic

infection can result in much severe and long lasting economic losses by

its own persistent infection and simultaneous secondary infection with

other pathogens, even if the chronic case is not severe by itself.

In particular, the clinical development of PRRS or swine enzootic

pneumonia makes the affected pig to be very sensitive to secondary infections of other respiratory diseases, for example, Glasser's disease,

pleuropneumonia, swine influenza, Streptococcus spp. and Salmonella

cholerasuis. And, if the porcine respiratory disease progresses to PMWS

and PRDC, it results in poor daily weight gain, low feed efficiency, high

post-weaning death and more respiratory symptoms in piglets and growing

pigs. Thus, there is the need for developing a method of preventing

these outstanding porcine respiratory diseases which may result in severe

economic losses in a farm. Also, the need for practically preventing PMWS and PRDC has been increased. However, there has been no appropriate

vaccine to meet fully these needs. In this regard, we, inventors, have studied to find a method of

preventing these outstanding porcine respiratory diseases at the same

time with one solution effectively. And we completed this invention by

designing a mixed vaccine containing three kinds of antigens, and by

assaying safety and antibody formation in an animal model.

DETAILED DESCRIPTION OF THE INVENTION

This invention is for providing a mixed (combined) vaccine for

three (3) types of porcine respiratory diseases. More specifically, this

invention is for providing an inactivated mixed vaccine for preventing

swine Glasser's disease in pigs, swine enzootic pneumoniae and porcine

reproductive and respiratory syndrome(PRRS) .

Other object of this invention is to provide an inactivated mixed

vaccine for reducing the causes of development of the three types of

porcine respiratory diseases, PMWS and PRDC of a growing-finishing pig.

Other object of this invention is to provide a novel method of

detecting neutralizing antibody titer of PRRS using a Guinea pig when

preparing the mixed vaccine of this invention. In order to achieve the objects mentioned above, the inventors

designed an effective inactivated mixed vaccine having excellent immunity

which can prevent or reduce disease causes that make the starter and

grower to develop the three types of porcine respiratory diseases of

swine Glasser' s disease, swine enzootic pneumonia and PRRS, PMWS (Post- weaning Multisystemic Wasting Syndrome, and PRDC (Porcine Respiratory

Disease Complex). In this invention, the vaccine for Glasser's disease

was prepared by inactivating Haemophi lus parasuis S4 and S5 strains with

using formalin which were isolated from a piglet suffering from illness

in a domestic swine farm. The vaccine for swine enzootic pneumonia was

prepared by inactivating the Mycoplasma hyopneumoniae J/101 strain using

formalin. The vaccine for PRRS was prepared using MN-HS (Minnesota

University, Prof. Han Soo Joo, ATCC No.: VR2509) or CNV-1 [obtained from

Prof. Kim, Hyeon Su, Chungnam National University, Korea; Sequence

analysis of 0RF4 gene of porcine reproductive and respiratory syndrome

virus (PRRSV) Korean isolate CNV-1, Korean J. Vet Res (1999) 39(2):

294-300] from a swine lung macrophage cell, a STL (swine testis cell

line) or a A-72 cell (ATCC No.: CRL-1542). After culturing the infected

cells, it is isolated when about 80% cytopathic effect (CPE) is observed,

for preparing vaccine.

In the meantime, the typical Serum Neutralization (SN) test cannot

provide an antibody titer when an inactivated PRRS vaccine is injected to an antibody negative pig. In this regard, the inventors designed a novel

antibody titer assay system. That is, the inventors firstly determined

the correlations between a guinea pig and a pig, regarding vaccination

using the inactivated PRRS vaccine. Then, the inventors assayed the

neutralizing antibody titer using a guinea pig. Thus, the novel method

of detecting PRRS neutralizing antibody titer using a guinea pig resolves

the problems which cannot be overcome using a pig. Since it is very

difficult to obtain a pig which remains negative for PRRS virus (90.4%

positive in farms, and 45.1% positive for pigs), and since the

neutralizing antibody (IgG) to PRRS virus is presented at later stage

(after 4-5 weeks from vaccination), it is not easy to detect the

neutralizing antibody titer. In order to overcome the problems, the

inventors employed a guinea pig instead of a pig for detecting neutralizing antibody titer as mentioned above. Neutralizing titer to

PRRS virus was detected after injecting the mixed vaccine of this

invention to a guinea pig and a piglet. Neutralizing at 4^°C for 48 hrs

with a complement (containing guinea pig' s fresh-sera in 1 wt% ~ 5 wt%)

was confirmed as the most effective method.

The mixed vaccine composition of this invention preferably

comprises in a range of 20 - 30% (vol/vol) vaccine components. The

components and their preferable contents are as follows: Haemophilus parasuis S4 strain (0.D 0.4 - 0.5 at 410 nm), Haemophilus parasuis S5 strain (0.D 0.4 ~ 0.5 at 410 nm), Mycoplasma hyopneumoniae J/101 (0.D 0.

05 - 0.15 at 410 nm) and PRRS MN-HS (10⁵-⁰ ~ 10⁸ TCID₅₀ /ml) . After fully

mixing the vaccine components, the mixture was filtered using copper net,

and then the aluminum hydroxide gel 10 - 20% (vol/vol) was added to the

filtered mixture in order for the vaccine components to be adsorbed onto

the gel. Next, oil adjuvant (IMS 1313 NPR) 5-10 % (vol/vol) and EDTA

0.14 % (w/vol) were added to the mixture to obtain the mixed vaccine

composition.

In one embodiment, the safety of the mixed vaccine of this

invention was evaluated using a mouse, a guinea pig and a piglet. As a result, the mouse and the guinea pig survived 7 days following the

vaccination, and the piglet survived 14 days following the vaccination-.

In other embodiment, immunogenicity of the mixed vaccine of this

invention was determined. As shown in table 9, the mixed vaccine induced

antibody formation. In other embodiment, the detection of neutralizing antibody titer

to PRRS virus using a guinea pig was compared to using an antibody

negative pig. According to the results, using a guinea pig for detecting

antibody titer to PRRS virus provides more accurate antibody titer. In other embodiment, the activity of the mixed vaccine of this

invention was monitored every 3 months during storage in a refrigerator

for 21 months. The results demonstrated that the vaccine of this invention had been valid and had no problem during long-time storage

(table 15, 16, 17 and 18).

In other embodiment, the safety and the immunogenicity were

demonstrated in the piglets of farms. The vaccine of this invention

showed excellent safety and immunogenicity (table 19 and table 20).

In other embodiment, finished stage of marketed pigs, daily weight

gains and marketing rates of commercial pigs were evaluated for a group

of pigs given with the mixed vaccine of this invention and for a group of

pigs given with the conventional vaccines (table 21, 22, 23 and 24) in

three farms. As for 11-week-old pigs, the average weight of the pigs given with the mixed vaccine of this invention was in a range of 3.1-4.2

kg, which demonstrated that the weight gain rate of the pigs treated with

the mixed vaccine of this invention was higher than that of the pigs

treated with the conventional vaccine. And the marketing rate of the

pigs treated with the mixed vaccine of this invention was 10% higher than

that of the pigs treated with the conventional vaccine. Thus, the

average marketing time of the group treated with the mixed vaccine of

this invention was shortened by 10 days in comparison to the group

treated with the conventional vaccine. BRIEF DESCRIPTION OF DRAWINGS

Fig. 1 to Fig. 3 show examples of pneumonia scores.

Fig. 1 is an example of pneumonia, and shows percentage of each lobe for entire normal lung.

Fig. 2 is an example of pneumonia, and shows lung lesion having 12% of lobe score.

Fig. 3 is an example of pneumonia, and shows lung lesion having 20% of lobe score.

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

EXAMPLE

Example 1: Isolation and culturing virus

As for Glasser's disease in pigs, each of Hae ophi lus parasuis S4

and S5 strains (obtained from National Veterinary Research and Quarantine

Service) were incubated in TSB (Tryptic Soy Broth) medium including β-

NAD (β-Nicotinamide adenine dinucleotide) 45 βg/mi , yeast extract 0.5%

and sucrose 0.5%. Then the incubated strains were isolated and were in

freeze-drying and cryopreservation at -80°C. A Mycoplasma hyopneumoniae

J/101 strain (obtained from National Veterinary Research and Quarantine Service), which causes swine enzootic pneumonia, was delivered in

mycoplasma medium (table 1) and was incubated. After the isolation, the incubated strain was in freeze-drying and cryopreservation at -80^°C.

Next , the Mycoplasma hyopneumoniae J/101 strains were transferred to

mycoplasma medium (table 1) in 1/3 volume of the medium, and incubated

for 7 to 10 days until the color of the medium was changed to yellow

color. The pathogen of porcine reproductive respiratory syndrome of MN-

HS virus (Minnesota University, Prof. Han Soo Joo, ATCC No.: VR2509) or

CNV-1 (obtained from Prof. Kim, Hyeon Su, Chungnam National University,

Korea) was inoculated and incubated in a swine lung macrophage cell, a

STL (swine testis cell line), or a A-72 cell (ATCC No.: CRL-1542) (table

2, cell culture medium) until 80% CPE appeared, followed by isolating the

incubated strains. Subsequently, the isolated strains were in freeze-

drying and cryopreservation at -80^°C in order to use them as vaccine

components. Next, the swine lung macrophage cell, the STL cell, or the

A-72 cell was suspended in a cell culture medium (table 2). The cell

suspension was incubated at 37^°C for 1-3 days, following the allocation

of the suspension into bottles. The cultured solution was removed when

the cultured cell formed a layer. Then, 10³'⁰ TCID₅₀/ml (50% tissue

culture infection dose) of the isolated MN-HS virus was transferred in a

virus culture medium (table 3) and was cultured with a rotary cell

culture system at 37^°C.

Table 2. Components of a cell culture medium (total volume: 1,000 ml)

Table 3. Components of a virus culture medium (total volume: 1,000 ml)

Example 2: Isolation and inactivation

As for Glasser's disease in pigs, respective seed culture solutions

of example 1 were delivered in a TSB medium including β-NAD (β-

Nicotinamide adenine dinucleotide) 45 μg/ml , yeast extract 0.5% and were

incubated at 37^°C for 12 hrs. Then, the incubated strains were isolated

and concentrated, and were inactivated using 0.3% formalin for 72 hrs.

As for swine enzootic pneumonia, Mycoplasma hyopneumoniae J/101 strains

were delivered in a Mycoplasma culture medium, and were incubated at 37^°C

for 7-10 days. Then, the incubated strains were isolated and

concentrated, and were inactivated using 0.2% formalin at room

temperature for 72 hrs. As for PRRS virus, MN-HS or CNV-1 was inoculated

to a pig' s lung macrophage cell, a STL (swine test is cell line) cell or

a A-72 cell, and incubated at 37^°C for 3-5 days using a rotary incubation

system. When about 80% CPE following the inoculation of virus was

observed, the virus was isolated and inactivated at room temperature for

48 hrs using 0.1% formalin. Regarding the Glasser's disease and swine

enzootic pneumonia, the inactivated strains were diluted appropriately.

Then, O.D. values (at 410 nm) for the diluted strains were detected using

UV spectrophotometer. The inactivated PRRS virus was 1/10 diluted, and

0.1 ml of the diluted solution was inoculated to a pig' s lung macrophage

cell, a STL (swine testis cell line) cell or an A-72 cell, followed by

incubating the cell at 37^°C for 72 hrs. The virus content was determined with the appearance of CPE following the PRRS virus inoculation. The

virus content was over 10^6,0 TCIDso/mβ.

Example 3: Preparation of inactivated mixed vaccine Strain isolation, disease causing agent isolation and inactivation

were carried out for the respective infection culture solutions using the

three kinds of antibodies of the example 2. The total contents of

vaccine components were 30% (vol/vol) of the whole mixed vaccine

composition. And the O.D. values of the respective vaccine components

were as follows: Haemophilus parasuis S4 (at 410 nm, O.D 0.45), Haemophilus parasuis S5 (at 410 nm, O.D 0.45), Mycoplasma hyopneumoniae J/101 (at 410 nm, O.D 0.1) and PRRS MN-HS (10⁶'°TCID₅₀ /ml) . After mixing

the vaccine composition, it was filtered with copper net. Then, the

filtered composition was adsorbed using aluminum hydroxide gel 20%

(vol/vol) followed by additionally adding oil adjuvant (IMS 1313 NPR) 5%

(vol/vol) and EDTA 0.14% (w/vol), and was mixed at 1,000 rpm for 30

minutes to provide a mixture. Next, the defined amount of mixture was

allocated to bottles and was stored at 2~7^°C in a cold room. The

obtained mixed vaccine composition formed two separated layers of a rose

pink colored liquid layer and a precipitation layer, wherein the layers

were easily mixed to provide homogenized solution when they were shaken.

The composition did not have any taste and smell. The vaccine sample in each lot was transferred to Thioglycollate medium (Thio), Nutrient agar

(NA) and Nutrient broth (NB) , respectively, and was incubated at 22^°C for

7 days. Additionally, the vaccine sample in each lot was transferred to

Thioglycollate medium (Thio), Nutrient agar (NA) and Nutrient broth (NB),

respectively, and was incubated at 37^°C for 7 days. During the

incubation, no contamination of the medium was recorded (table 4). The

respective vaccine samples were used for the following experiments. The

contents of formalin in the respective lots were in a range of 0.19 -

0.20%(vol/vol) (table 5).

Table 4. Contamination of various germs

* - : No contamination

Table 5. The contents of formalin

Example 4: Evaluation of safety in an animal model

(1) Safety test in a mouse

For this experiment, 10 mice weighing 13 g and 10 mice weighing 15 g were used. Respective vaccine samples (0.5 ml, lot no. 1-3) of example 3 were delivered intraperitoneally to 5 mice weighing 13 g and 5 mice weighing 15 g. And respective vaccine samples (0.5 ml, lot no. 1-3) of example 3 were delivered subcutaneously to 5 mice weighing 13 g and to 5 mice weighing 15 g. The vaccinated mice were monitored in comparison to 5 untreated control mice. All the mice, which were injected with the mixed vaccine of this invention, survived during the monitoring period (table 6).

Table 6. Safety test for the mixed vaccine using mice

(2)Safety test in a guinea pig

Respective 12 guinea pigs weighing 300 g and 350 g were used in this experiment. Respective 4 guinea pigs weighing 300 g and 4 guinea pigs weighing 350 g were injected intramuscularly by the respective vaccine sample of example 3 (0.1 ml). And respective 4 guinea pigs weighing 300 g and 4 guinea pigs weighing 350 g were injected intradermally by the respective vaccine sample of example 3 (0.1 ml). And

respective 4 guinea pigs weighing 300 g and 4 guinea pigs weighing 350 g

were injected subcutaneously by the respective vaccine sample of example

3 (0.1 ml). The vaccinated mice were monitored in comparison to 4

untreated guinea pigs for controls. All the guinea pigs survived during

the monitoring period (table 7). Table 7. Safety test using guinea pigs

(3) Safety test in a piglet 18 one-week-old piglets, which were antibody negative to

Haemophilus parasuis, Mycoplasma hyopneumoniae and PRRS virus, were used in this experiment. Five piglets were injected through neck muscle using

the mixed vaccine (2.0 ml, Lot no. 1) of the example 3. Likewise, five piglets were injected through neck muscle using the mixed vaccine (2.0 ml,

Lot no. 2) of the example 3. Further, five piglets were injected through

neck muscle using the mixed vaccine (2.0 ml, Lot no. 3) of the example 3.

The piglets injected with the mixed vaccine of this invention and the 3

naive piglets for controls were monitored for 14 days. As results, no

piglets showed clinical symptom, for example, anorexia, respiratory

symptom (e.g. cough and difficult breathing), diarrhea, emesis and

neurological symptom (see table 8). And they maintained normal body

temperature in a range of 38.7 - 39.7 ^°C .

normal Example 5: Immunogenecity test

18 one-week-old piglets, which were antibody negative to Haemophilus parasuis, Mycoplasma hyopneumoniae and PRRS virus were used

in this experiment. Five piglets were injected through neck muscle

using the mixed vaccine (1.0 ml, Lot no. 1) of the example 3. Likewise,

five piglets were injected through neck muscle using the mixed vaccine

(1.0 ml, Lot no. 2) of the example 3. Further, five piglets were injected

through neck muscle using the mixed vaccine (1.0 ml, Lot no. 3) of the

example 3. 2 weeks after the first injection, the respective piglets

were injected with the same vaccine (2.0 ml) as the one used in the first injection through near ear muscle. 4 weeks after the second injection,

blood was collected from the piglets inoculated with vaccine and from the

two piglets untreated, in order to assay antibody titers. ELISA antibody

titer for Haemophi lus parasuis increased 80-160 fold after 2 weeks from

the first injection, and increased 320-1,280 fold after 4 weeks from the

second injection, respectively. Not more than 10 fold antibody titer was

detected for the untreated pigs for controls. ELISA antibody titer for

Mycoplasma hyopneumoniae increased 80-320 fold after 2 weeks from the

first injection, and increased 640-2,560 fold after 4 weeks from the second injection, respectively. Not more than 10 fold antibody titer was

detected for the untreated pigs for controls. Antibody titer to PRRS

virus was not more than 2 fold after 2 weeks from the first injection, and was in a range of 4-8 fold after 4 weeks from the second injection, respectively. Not more than 10 fold neutralizing antibody titer was detected for the untreated control pigs (table 9).

Example 6: Detection of neutralizing antibody titer in a guinea pig

12 guinea pigs (300-350 g) were used in this experiment. 10 of them were inoculated intramuscularly using 1 ml of the mixed vaccine of this invention, and were additionally injected with the same amount of the mixed vaccine after three weeks from the first injection. 2 of the guinea pigs were used as controls. 2 weeks after the second injection,

blood was gathered from the pigs in order to detect neutralizing antibody

titers. 8 of 3-week-old piglets were obtained from a farm, wherein the

farm was confirmed as antibody negative. 6 of them were injected through

neck muscle with the vaccine sample (2 ml), and were additionally

injected after three weeks from the first injection. And two of them

were used as controls. The blood samples of pigs were collected before the first injection, after three weeks from the first injection, after 2

weeks from the second injection, and after 4 weeks from the second

injection, respectively. The neutralizing antibody titers were detected

using the collected blood, and the results were compared to the results

of the conventional method as mentioned below. That is, the conventional

method included, blood serum inactivation at 56^°C for 30 min., 1/2

dilution of the blood serum in a 96 well microplate containing 50 μl of

cell culture medium (α-MEM) disclosed in table 3 of the example 1,

addition of 50 μl PRRS virus culture solution (200 TCIDso/O.lml) into the

diluted serum and neutralizing it at 37^°C for 1 hour, additional addition

of 50 μl medium comprising 3% FBS which contained 250,000 of PRRS virus

sensitive cells (MARC-145, MA-104), incubation of the mixture at 37^°C in

5% C0₂ incubator for 5 days, and detection of CPE in order to assay the

neutralizing antibody titers. The neutralizing antibody titer assays

were carried out at 37^°C for 1 hour and at 4^°C for 48 hours using guinea pigs and pigs, and the results were compared to each other (table 10 and

table 11). In case of neutralization at 4°C for 48 hours, media having

different amount of guinea pig' s fresh-serum (none, serum 1 wt%, serum 5

wt%) were added as complements. Further, reproducibility of the result

was confirmed by carrying out neutralization at 4^°C for 48 hours with a

complement (1% fresh serum of guinea pig). The neutralizing antibody

titer was also assayed using an antibody negative piglet at 4^°C for 48

hours with a complement (1 wt% fresh serum of guinea pig), which was

different from the conventional method. The following tables 10 to 14

disclose the results of this experiment. From the results, it is

understood that the guinea pig can be used as an excellent specimen for

assay neutralizing antibody titer.

Table 10. Assay of neutralization antibody titer according to neutralization time

* 37^°C, 1 hour; ** 4^°C , 48 hours

Table 11. Assay of neutralization according to the amount of complement

* None, ** serum, *** 5% serum Table 12. Complement (1% serum), at 4^°C for 48 hrs.

* tested by Sung-sik Yoo at Choongang Vaccine Laboratory Co., Ltd. ** tested by Yeon-sil Lee at Choongang Vaccine Laboratory Co., Ltd. *** tested by Hyeok~j in Kwon at Choongang Vaccine Laboratory Co., Ltd. Table 13. Conventional method for assaying serum neutralizing antibody titer for PRRS virus in antibody negative pigs.

Table 14. Serum neutralizing antibody titer assay at 4^°C for 48 hrs in antibody negative pigs (1% serum)

Example 7' long-time storage test

The mixed vaccine of this invention was monitored while being stored in a refrigerator for a long time. The vaccine sample was stored in a range of 2~7^°C in a dark cold place. While storing the vaccine, 1.0 ml of it was injected through neck muscle to 1-week-old healthy piglets, which were antibody negative to Haemophilus parasuis, Mycoplasma hyopneumoniae and PRRS virus, every 3 months from the date of preparation for 21 months. After 2 weeks from the first injection, 2.0 ml of vaccine sample was injected to the piglets. After 4 weeks from the second0 injection, sera was collected and ELISA antibody titer (H. parasuis and M. hyopneumoniae) and neutralizing antibody titers were assayed. The immunogenicity was effective and the stability was satisfactory until 21 months (table 15, 16, 17 and 18).

Table 15. Stability test for the mixed vaccine ( ~ 3 months)

Table 18. Stability test for the mixed vaccine (18-21 months)

Example 8: Field test

(1) Safety test in a piglet In order to test safety in a piglet, 330 1-week-old healthy piglets were selected. 30 piglets of 330 piglets were used as controls. 300 piglets were inoculated with the mixed vaccine of this invention (2.0 ml) through neck muscle, and were monitored in comparison to the untreated piglets for 14 days. No piglets showed clinical symptom, for example, anorexia, respiratory symptom (e.g. cough and difficult breathing), 0 diarrhea, emesis and neurological symptom (see table 19). The body temperatures of the vaccinated 30 piglets (respective 10 piglets of 3 kinds of vaccine samples) and 10 piglets for controls were detected. All the piglets showed normal temperatures (table 19). It demonstrates that the mixed vaccine of this invention is safe in target animals. Table 19. Safety test in a piglet

(2) Immunogenecity test in a piglet

31 1-week-old piglets, which were antibody negative to Haemophilus parasuis, Mycoplasma hyopneumoniae and PRRS virus were used in this experiment. 3 types of the mixed vaccine samples (1.0 ml) were delivered to 28 piglets through neck muscle. After 2 weeks from the first injection, the piglets were additionally injected with the respective 3 types of vaccine samples (2.0 ml) through neck muscle. After 4 weeks from the second injection, the sera of the piglets including untreated piglets was collected and antibody titers were detected. ELISA antibody titer to Haemophilus parasuis and Mycoplasma hyopneumoniae was detected. The neutralizing antibody titer was detected for PRRS virus. ELISA antibody titer for Haemophilus parasuis increased 80-160 fold after 2 weeks from the first injection, and increased 320-1,280 fold after 4 weeks from the second injection, respectively. ELISA antibody titer for Mycoplasma hyopneumoniae increased 80-320 fold after 2 weeks from the

first injection, and increased 640-2,560 fold after 4 weeks from the

second injection, respectively. Not more than 10 fold antibody titer was

detected for the untreated control pigs. Antibody titer to PRRS virus

was not more than 2 fold after 2 weeks from the first injection, and was

in a range of 4-8 fold after 4 weeks from the second injection,

respectively. Not more than 2 fold antibody titer was detected for the

untreated control piglets.

Comparative example

Comparative experiment was carried out for 6 months at 3 farms

using the mixed vaccine of this invention, wherein the pigs of the farms

had been treated with other mixed vaccine or monovalent antigen vaccine.

Marketing age, marketing rate, and average body weight were monitored for

this purpose. Table 21. Farms and vaccine

Table 22. The changes of the average body weight of this invention' s

vaccine treating group and the conventional vaccine treating group

Table 23. The number of pigs and average marketing age

Table 24. Lung lesion scores of the mixed vaccine treated group and the

conventional vaccine treated group

As shown in table 21, the piglets inoculated with the mixed

vaccine of this invention weigh in an average range of 1.5-2.0 kg more

than those vaccinated with the conventional vaccine from 7 week old. And

the piglets inoculated with the mixed vaccine of this invention weigh in

an average range of 3.1-4.2 kg more than those vaccinated with the

conventional vaccine from 11 week old. Referring to table 22, the group

injected with the mixed vaccine of this invention showed increased

marketing rate of 92.2-94.9%, while that of the group injected with the

conventional vaccine was 83.6-84.4%. Thus, the average marketing age was

shortened about 8-13 days in comparison to the group treated with the conventional vaccine. According to table 23, the lung lesion scores of

the group treated with the conventional vaccine were within in range of

30.4-33%, while the scores for the group treated with the mixed vaccine

of this invention were within in a range of 5.5-8.2%. The detection of

lung lesion score was carried out in accordance with the known method

(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.) (see fig. 1).

INDUSTRIAL APPLICABILITY

Thus, the mixed vaccine of this invention can effectively prevent

the infection of outstanding swine respiratory diseases of Gasser's

disease, Swine enzootic pneumonia and Porcine Reproductive and

Respiratory Syndrome. Further, the mixed mixed vaccine of this invention

provides a practical method of preventing progress of the porcine

respiratory disease to PRDC (Porcine Respiratory Disease Complex) and

PMWS (Post-weaning Multisystemic Wasting Syndrome) due to neonatal

mortality, atrophy and decrease of daily weight gain.

Claims

We claim,

1. An inactivated mixed vaccine for preventing Glasser's disease in pigs, Swine enzootic pneumonia and Porcine Reproductive & Respiratory Syndrome (PRRS), and for preventing disease to progress to Post-weaning Multisystemic Wasting Syndrome (PMWS) and Porcine Respiratory Disease Complex (PRDC), which comprises: Haemophilus parasuis S4 & S5 (Korean National Veterinary Research and Quarantine Service) (at 410 nm, O.D 0.4 ~ 0.5) which is inactivated using formalin; Mycoplasma hyopneumoniae J/101 (Korean National Veterinary Research and Quarantine Service) which is inactivated using formalin (at 410 nm, O.D 0.05 ~ 0.15);

MN-HS (ATCC No.: VR2509) or CNV-1 (Prof. Kim, Hyeon Su, Chungnam National University, Korea) (10⁵'° - 10⁸'⁰ TCID₅₀ / l) , wherein MN-HS (ATCC No.: VR2509) or CNV-1 virus is incubated in a pig lung macrophage, a STL (swine test is cell line) or an A-72 cell (ATCC No.: CRL-1542) until 80% CPE is observed, followed by isolation and inactivation; and pharmaceutically acceptable excipients.

2. A method of preparing an inactivated mixed vaccine for preventing Glasser's disease in pigs, swine enzootic pneumonia and Porcine Reproductive & Respiratory Syndrome (PRRS), and for preventing disease to progress to Post-weaning Multisystemic Wasting Syndrome (PMWS) and Porcine Respiratory Disease Complex (PRDC), which compr ises : incubating and inactivating Haemophi lus parasuis S4 & S5 and Mycoplasma hyopneumoniae J/101 using formalin; incubating MN-HS or CNV-1 virus using a pig' s lung macrophage cell, STL (swine test is cell line) or an A-72 cell, isolating the incubated virus when 80% CPE occurs and inactivating the isolated virus with formalin; detecting ELISA antibody titer to Haemophi lus parasuis S4 & S5 and Mycoplasma hyopneumoniae J/101; detecting neutralizing antibody titer to MN-HS or CNV-1 virus using guinea pig' s fresh serum as a complement; and mixing the inactivated vaccine components with pharmaceutically acceptable excipients.

3. The method of claim 2, wherein the detection of neutralizing antibody titer to virus using guinea pig' s serum is carried out with a guinea pig or a piglet, following the neutralizing antibody at 4^°C for 48 hrs with a complement of guinea pig' s fresh-serum

1-5 wt%. he method of claim 2 or claim 3, wherein MN-HS or CNV-1 virus is

incubated in a STL (Swine test is cell line) cell or A-72 cell not

in a simian cell.