# Patent

## Title
Mycoplasma vaccine, method of making, and application thereof

## Abstract

The present invention provides a mycoplasma vaccine, its preparation and application thereof. The foregoing mycoplasma vaccine comprises inactivated Mycoplasma hyorhinis ATIT-7 only or the mixture of inactivated Mycoplasma hyorhinis ATIT-7 and inactivated Mycoplasma hyopneumoniae, which effectively prevents the infection of swine enzootic pneumonia in pigs.

## Background
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a vaccine prepared from Mycoplasma hyorhinis isolated from the lung lesions of pigs infected with enzootic pneumonia and the application of the foregoing vaccine.
2. Description of Related Art
Swine enzootic pneumonia is a chronic disease characterized by high infection rate and low mortality that infects 25 ̃93% of swine herds and is positive in 28 ̃80% of lung tissues of carcasses. The growth efficiency of infected pigs is reduced by 14 ̃16%. In high-density rearing environment that is poorly ventilated and moist with wildly changing climate, the incidence and spread of swine enzootic pneumonia can rise to an alarming level, resulting in lower feed conversion, regarded growth, inflammatory reaction and immunosuppression in pigs. This disease is often accompanied by secondary infection of opportunistic pathogens, such as Actinobacillus pleuropneumoniae, Pasteurella, and Streptococcus suis, leading to serious economic loss and becoming one of important reasons for the cost increase of the pig industry.
The strategic approach to the prevention of swine enzootic pneumonia in Taiwan is to add antibiotic in the feed. But long-term use of antibiotic is prone to produce resistant strains and leads to the problem of residual antibiotic in the meat products, which poses significant health issue. Field experience also shows the preventive effect of feeding animals with drugs is not as ideal as expected.
There are three commonly seen mycoplasma in pigs, which are Mycoplasma hyopneumoniae (M. hyopneumoniae), Mycoplasma hyorhinis (M. hyorhinis), and M. flocculate. M. hyopneumoniae is the important causative organism of swine enzootic pneumonia (SEP); M. hyorhinis is the etiological agent of polyserositis and arthritis; M. flocculate has not been shown to cause diseases. In the past, all SEP incidences were caused by M. hyopneumoniae. But recently it is found that such disease is caused by either M. hyorhinis alone or the combination of M. hyorhinis and M. hyopneumoniae. In Taiwan, mycoplasma isolated from the lung lesions of pigs infected with SEP was only M. hyopneumoniae prior to 1996. In the case reports in other countries, M. hyorhinis was primarily isolated from the synovial fluid of pigs infected with arthritis, which did not cause SEP and was not considered an important pathogen for swine diseases. Taiwan never isolated this pathogen in the past. But starting in 1996, the Mycoplasma Laboratory of Animal Technology Institute Taiwan finds mycoplasma isolated from the lung lesions of pneumonia-infected pigs to be M. hyorhinis in more incidences as confirmed by antibody binding reaction using Western blotting and comparison with ATCC standard strains.
The Animal Technology Institute Taiwan provides mycoplasma isolation and identification service to pig farms around the country, and sees higher and higher incidence of M. hyorhinis isolates from pneumonia cases. In the 242 cases in 2001 and 205 cases in 2002, the M. hyopneumoniae infection rate dropped from 46.8% in 2001 to 15.8% in 2002, while that of M. hyorhinis rose from 65.5% in 2001 to 79.2% in 2002. The infection rate of the mixture of M. hyopneumoniae and M. hyorhinis was 14.4% in 2001 and 15% in 2002. These figures indicate rapidly rising M. hyorhinis infection in swine pneumonia cases in Taiwan and rapidly dropping infection rate of M. hyopneumoniae, while infection rate of the mixture of the two remains steady. It also indicates that M. hyorhinis is gradually replacing M. hyopneumoniae as the most significant pathogen of SEP. The past belief was that M. hyopneumoniae was the only species among mycoplasma to cause SEP. This is not the situation now. In the isolation cases described above, there was one pure M. hyorhinis infection case in 2001, and five such cases in 2002, suggesting M. hyorhinis alone could elicit SEP.
Field experience shows that the chance of reinfection with the same mycoplasma species is relatively low, indicating good innate immunity of the pigs against such pathogen. Thus using vaccination as a means of disease prevention is a viable approach. Given the weak cross reaction between the antigens of M. hyorhinis and M. hyopneumoniae, it is found in pig farm survey on vaccination that pigs administered with M. hyopneumoniae vaccine were not effectively protected against the infection of M. hyorhinis. For pigs infected with both mycoplasma species, the effect of administering M. hyopneumoniae vaccine or M. hyorhinis vaccine alone was not satisfactory. Only vaccine containing the mixture of both mycoplasma antigens provides adequate protection. Thus developing vaccine containing M. hyorhinis or both M. hyorhinis and M. hyopneumoniae is a pressing task.
SUMMARY OF THE INVENTION
For the prevention of swine enzootic pneumonia, the present invention provides a mycoplasma vaccine, comprising at least an effective amount of inactivated M. hyorhinis ATIT-7. The foregoing M. hyorhinis has been preserved at the Culture Collection and Research Center of the Food Industry Research and Development Institute (No. BCRC910223) since May 8, 2003. This isolate was also deposited in the American Type Culture Collection (ATCC, 10801 University Blvd., Manassas, Va. 20110-2209, USA) under the Budapest Treaty on Jun. 9, 2006, and was given the ATCC Accession No. PTA-7651. Besides containing M. hyorhinis ATIT-7, the foregoing mycoplasma vaccine may further include an effective amount of inactivated M. hyopneumoniae, wherein the concentration of M. hyopneumoniae in the vaccine is 60 ̃80% v/v and that of M. hyorhinis ATIT-7 is 40 ̃20% v/v.
The mycoplasma vaccine may further contain an adjuvant or diluent, wherein the inactivated mycoplasma fluid (containing only M. hyorhinis ATIT-7 or the mixture of M. hyorhinis ATIT-7 and M. hyopneumoniae) comprises 50 ̃75% v/v of the vaccine composition, and the adjuvant comprises 50 ̃25% v/v.
The mycoplasma vaccine may be administered subcutaneously or intramuscularly to the animal.
The M. hyorhinis ATIT-7 is cultured until its O.D.550 reaches the level of 0.14 to 0.33, and M. hyopneumoniae is cultured until its O.D.550 reaches the level of 0.08 to 0.16.
The present invention also relates to a method for the preparation of mycoplasma vaccine, comprising the steps of: culturing M. hyorhinis ATIT-7 in vaccine culture medium; inactivating the harvested M. hyorhinis ATIT-7 with formalin; and letting the culture stand under 2-8° C. to continue the inactivation for 16 to 72 hours. M. hyorhinis ATIT-7 is cultured until its O.D.550 reaches the level of 0.14 to 0.33 with viable count of higher than 109 CCU/mL. The concentration of said formalin is preferably between 0.1 and 0.5%, and more preferably between 0.1 to 0.2%.
The vaccine culture medium for cultivating M. hyorhinis comprises 500 ml of Hank's solution, 12,000 ml of distilled water, 82 g of Bacto brain heart infusion (Difco), 87 g of Bacto PPLO broth, 600 ml of yeast extract, 45 ml of phenol red, 2.5 g of bacitracin, 2.5 g of penicillin or methicillin, and 1,500 to 5,000 g of inactivated porcine serum or inactivated horse serum.
The method for preparing M. hyorhinis vaccine can further contain the steps of: culturing M. hyopneumoniae in a vaccine culture medium; inactivating harvested culture fluid with formalin; and letting the culture stand under 2-8° C. to continue the inactivation for 16 to 72 hours; admixing the resulting inactivated M. hyopneumoniae with aforesaid inactivated M. hyorhinis. The concentration of said formalin is preferably between 0.1 and 0.5%, more preferably between 0.1 to 0.2%. M. hyopneumoniae is cultured until its O.D.550 reaches the level of 0.08 to 0.16 with viable count of higher than 109 CCU/mL.
The vaccine culture medium for cultivating M. hyopneumoniae comprises 500 ml of Hank's solution, 12,000 ml of distilled water, 82 g of Bacto brain heart infusion (Difco), 87 g of Bacto PPLO broth, 600 ml of yeast extract, 45 ml of phenol red, 2.5 g of bacitracin, 2.5 g of penicillin or methicillin, and 1,500 to 5,000 g of inactivated porcine serum.
The present invention further provides a pharmaceutical composition for the prevention of mycoplasma infection, comprising an effective amount of the aforesaid mycoplasma vaccine and a pharmaceutically acceptable carrier.
The present invention also relates to a M. hyorhinis strain ATIT-7 capable of infecting swine and causing pneumonia, wherein said strain has been preserved at the Culture Collection and Research Center of the Food Industry Research and Development Institute (No. BCRC 910223) since May 8, 2003.
The present invention also provides a strain collection method, comprising the steps of: isolating M. hyorhinis strain ATIT-7 from the lung lesions of pigs infected with pneumonia; culturing said strain in culture medium under 35 ̃38° C. for 16 to 24 hours to obtain viable organism count of higher than 109 CCU/mL. The culture medium for cultivating M. hyopneumoniae comprises 500 ml of Hank's solution, 12,000 ml of distilled water, 82 g of Bacto brain heart infusion (Difco), 87 g of Bacto PPLO broth, 600 ml of yeast extract, 45 ml of phenol red, 2.5 g of bacitracin, 2.5 g of penicillin or methicillin, and 1,500 to 5,000 g of inactivated porcine serum or inactivated horse serum.
The M. hyorhinis strain ATIT-7 has been preserved at the Culture Collection and Research Center of the Food Industry Research and Development Institute (No. BCRC 910223) since May 8, 2003.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A shows the production of M. hyorhinis antibody in mice vaccinated with M. hyorhinis bacterin and bacterin containing the mixture of M. hyorhinis and M. hyopneumoniae. 
FIG. 1B shows the production of M. hyopneumoniae antibody in mice vaccinated with M. hyorhinis bacterin and bacterin containing the mixture of M. hyorhinis and M. hyopneumoniae. 
FIG. 2A shows the production of M. hyorhinis antibody in piglets vaccinated with M. hyorhinis bacterin and bacterin containing the mixture of M. hyorhinis and M. hyopneumoniae on days 1, 15, and day 29 respectively.
FIG. 2B shows the production of M. hyopneumoniae antibody in piglets vaccinated with M. hyorhinis bacterin and bacterin containing the mixture of M. hyorhinis and M. hyopneumoniae on days 1, 15, and day 29 respectively.
FIG. 3A-D observes the pathological changes of the lungs of piglets following immunoresistance test, in which A, B, C, D represents Group 1, 2, 3, and 4 respectively; Group 1 was vaccinated with M. hyorhinis bacterin and challenged with virulent M. hyorhinis; Group 2 was vaccinated with M. hyorhinis+M. hyopneumoniae bacterin and challenged with virulent M. hyorhinis and M. hyopneumoniae; Group 3 was not vaccinated but challenged with virulent M. hyorhinis; and Group 4 was not vaccinated and not challenged with mycoplasma. 



DETAILED DESCRIPTION OF THE INVENTION
The features and advantages of the present invention are further depicted with the illustration of examples.
EXAMPLE
Preparation of Vaccine
1. Vaccine Strain
PRIT-5 is a M. hyopneumoniae strain disclosed in another Taiwanese patent of the applicant filed on Apr. 24, 1990 and approved on Apr. 21, 1991. PRIT-5 strain has been preserved at the Culture Collection and Research Center of the Food Industry Research and Development Institute (No. CCRC910045) since Mar. 14, 1996. ATIT-7 is a M. hyorhinis strain isolated from lung lesions of infected pigs, which is found to proliferate very fast in culture medium; its viable count could reach over 109 CCU/mL after growing in culture medium under 35 ̃38° C. for 16 hours, while the count of other M. hyorhinis strains fell in the range of 108 ̃109 CCU/mL. The ATIT-7 strain has been preserved at the Culture Collection and Research Center of the Food Industry Research and Development Institute (No. BCRC910223) since May 8, 2003.
2. Preparation of Culture Medium
The culture medium for preparing the vaccine is formulated as follows:

















Hank's solution
500
ml



Distilled water
12,000
ml



Bacto brain heart infusion
82
g



Bacto PPLO broth
87
g



Yeast extract
600
ml



Phenol red
45
ml



Bacitracin
2.5
g



Penicillin or methicillin
2.5
g



Porcine serum
1,500 to 5,000
g










    
    
        (The porcine serum is first inactivated under 56° C. for 30 minutes one to two times)
    
    


The formulated culture medium has pH adjusted to 7.4 ̃7.6 and is then filtered with 0.2 μm Millipore.
3. Preparation of Vaccine
Culture and Treatment of M. Hyorhinis Vaccine
Let M. hyorhinis strain ATIT-7 grow in vaccine culture medium which is placed in 37° C. shaking incubator for 8-24 hours. Harvest the culture when the bacterial fluid turns yellow. Use photoelectric colorimeter to measure the O.D.550, which must reach 0.14 to 0.33, and the viable-organism count which must be higher than 109 CCU/mL. Add 0.1 to 0.2% formalin to the harvested culture. After carrying out inactivation in 37° C. shaking incubator for 1 hour, place the bacterial fluid under 2 ̃8° C. to continue inactivation for 16 to 72 hours. Mix the formalin-treated bacterial fluid with adjuvant (50 ̃75% v/v of bacterial fluid and 50 ̃25% v/v of formalin). Agitate the mixture with agitator for 15 minutes. The resulting vaccine is stocked under 4° C. for future use. Each dose of the vaccine is 2 ml with each ml containing about 2×109 ̃2×1010 CCU.
Culture and Treatment of M. Hyorhinis and Mixture Vaccine
Let strain PRIT-5 and ATIT-7 grow in vaccine culture medium which is placed in 37° C. shaking incubator for 36-72 hours (PRIT-5) and 8-24 hours (ATIT-7) respectively. Harvest the culture when the bacterial fluid turns yellow. Use photoelectric colorimeter for measurement. The O.D.550 of PRIT-5 must reach 0.08 to 0.16, and its viable-organism count must be higher than 109 CCU/mL; The O.D.550 of ATIT-7 must reach 0.14 to 0.33, and its viable-organism count must be higher than 109 CCU/mL.
Add 0.1 to 0.2% formalin to the harvested ATIT-7 and PRIT-5 cultures respectively. After carrying out inactivation in 37° C. shaking incubator for 1 hour, place the bacterial fluid under 2 ̃8° C. to continue inactivation for 16 to 72 hours. Admix the formalin-treated ATIT-7 bacterin and formalin-treated PRIT-5 bacterin by the respective ratio of 40 ̃20% v/v and 60 ̃80% v/v into a bacterin mixture.
Admix the bacterin mixture with adjuvant (50 ̃75% v/v of bacterin mixture and 50 ̃25% v/v of formalin). Agitate the mixture with agitator for 15 minutes. Each dose of the resulting vaccine is 2 ml with each ml containing about 2×109 ̃2×1010 CCU.
4. Use of Vaccine
Each piglet was given two or three intramuscular injections of the prepared vaccine at one dose each time. The first dose was administered at 1-3 weeks of age; the second dose was administered at 3-5 weeks of age; the third dose was administered at 5-7 weeks of age. The vaccine must be mixed well prior to use.
5. Vaccine Safety Test
(1) Safety test in mice: Obtain 40 BALB/c mice. Randomly assign 8 mice as control group and divide the remaining 32 mice into 4 test groups with 8 mice in each group. Group 1 were subcutaneously inoculated with 0.5 ml M. hyorhinis vaccine; Group 2 was subcutaneously inoculated with 0.5 ml mixture vaccine (mixture of M. hyorhinis and M. hyopneumoniae bacterins); Group 3 received intraperitoneal inoculation of 0.5 ml M. hyorhinis vaccine; and Group 4 received intraperitoneal inoculation of 0.5 ml mixture vaccine. The mice were observed for 14 days after vaccination. All mice survived and no adverse reaction was observed.
(2) Safety test in piglets: Pick 15 one-week old piglets. Randomly divide the piglets into 5 groups with 3 heads per group. Group 1 was administered with one dose of M. hyorhinis vaccine intramuscularly on the side of neck; Group 2 received 5 doses of M. hyorhinis vaccine intramuscularly on the side of neck; Group 3 was vaccinated with 1 dose of mixture vaccine intramuscularly on the side of neck; and Group 4 received 5 doses of mixture vaccine intramuscularly on the side of neck. All piglets survived and no adverse reaction was observed in subsequent 14 days of observation period.
6. Vaccine Efficacy Test
(1) Antibody titer assay in mice: Obtain 30 four-week old BALB/c female mice. Randomly divide them into 3 groups with 10 mice in each group. Group 1 and Group 2 were subcutaneously vaccinated twice with M. hyorhinis vaccine and mixture vaccine respectively. Group 3 was the control group. In one week after the second vaccination, blood was collected from eye orbit under anesthesia. The collected blood was placed under room temperature for 1 hour and then placed under 4° C. overnight. The blood was then centrifuged under 1107×g for 30 minutes. After centrifugation, supernatant was removed, placed in a new centrifuge tube, and then subject to ELISA immunoassay. The results are as shown in FIG. 1. FIG. 1A shows the level of M. hyorhinis antibody produced in mice vaccinated with M. hyorhinis vaccine (Group 1) and mixture vaccine (Group 2); FIG. 1B shows the level of M. hyopneumoniae antibody in mice vaccinated with mixture vaccine (Group 2). It is clear that two administrations of M. hyorhinis vaccine or M. hyorhinis-M. hyopneumoniae mixture vaccine will boost the level of serum antibody and thus enhance the pig's immune reaction.
(2) Immunoresistance test in piglets: The purpose of resistance test is to compare the immunity of vaccinated and non-vaccinated piglets against mycoplasma infection. Obtain 12 3-week old piglets which were divided into 4 groups with 3 heads each. Group 1 was vaccinated with M. hyorhinis vaccine and then challenged with virulent M. hyorhinis; Group 2 was administered with mixture vaccine and then challenged with virulent M. hyorhinis and M. hyopneumoniae; Group 3 (control group) was given PBS and challenged with virulent M. hyorhinis; and Group 4 (control group) was given PBS and challenged with PBS instead of virulent mycoplasma (see Table 1). The first vaccination was given on day 1, the booster shot was given on day 15, and the challenge was carried out on day 29. The piglets were sacrificed on day 50. Blood was collected three times prior to vaccination and challenge on days 1, 15, and 29 respectively, and then subjected to ELISA immunoassay. The results are as shown in FIG. 2. FIG. 2A shows that M. hyorhinis serum antibody was observed in both Group 1 and Group 2, and the antibody level peaked after the second vaccination (day 29). FIG. 2B shows the presence of M. hyopneumoniae serum antibody in Group 2 piglets vaccinated with mixture vaccine. Both graphs indicate rising mycoplasma serum antibodies in vaccinated piglets. After two doses of vaccines, Group 1 and Group 3 were challenged with virulent M. hyorhinis, while Group 2 was challenged with both virulent M. hyorhinis and M. hyopneumoniae, and Group 4 was challenged with PBS in place of mycoplasma as control. In three weeks after the challenge, the piglets were weighed and then euthanized and necropsied. The pathological changes of the lungs of necropsied piglets are shown in FIG. 3. FIG. 3A shows the lung from Group 1, FIG. 3B shows the lung from Group 2, and so on. The harvested lungs had lesion count and microorganism isolation with results depicted in Table 2. The average body weight of the vaccinated group and non-vaccinated group differed by nearly 15 kg, suggesting M. hyorhinis infection significantly retarded the growth of pigs, while the vaccinated groups were not affected. Based on the observation of lung lesions in FIG. 3 and lung lesion count as depicted in Table 2, it is found that the lungs of vaccinated groups and non-challenged group (Group 4, FIG. 3D) did not have lesions, while non-vaccinated and challenged group (Group 3, FIG. 3C) showed typical mycoplasma pneumonia lesion (at where black arrow is pointed at) with striking difference between the two.







TABLE 1







Vaccine Efficacy Test Design













No. of piglets
Day 1
Day 15

Day 50


Group
vaccinated
vaccination
vaccination
Day 29 challenge
necropsy















1
3
M. hyorhinis
M. hyorhinis
M. hyorhinis
—


2
3
M. hyorhinis +
M. hyorhinis +
M. hyorhinis +
—




M. hyopneumoniae
M. hyopneumoniae
M. hyopneumoniae


3
3
PBS
PBS
M. hyorhinis
—


4
3
PBS
PBS
PBS
—
















TABLE 2







Post-challenge Growth, Lung Lesion Count and M. hyopneumoniae


isolation











Body weight (kg)
Lung














Before
Before
Before
lesion
Mycoplasma


Group
vaccination
challenge
necropsy
count
isolation















1
4.3 ± 0.1
15.1 ± 0.5
37.2 ± 3.4
0
0/3


2
4.4 ± 0.2
14.9 ± 0.3
39.8 ± 1.5
0
2/3


3
4.0 ± 0.2
13.8 ± 0.3
24.1 ± 2.3
7.3 ± 5.0
2/3


4
3.9 ± 0.2
14.2 ± 0.3
34.2 ± 1.6
0
0/3









(3) Field test: To understand the ability of vaccine of the present invention to elicit protective immunity in the field, vaccines were provided to two pig farms; one had incidence of simple M. hyorhinis infection (herds of 1,300 pigs), and the other had incidence of M. hyorhinis and M. hyopneumoniae mixed infection (herds of 4,000 pigs). After vaccination, the pig farm that had simple M. hyorhinis infection saw the number of piglet death drop from 137 heads to 50 heads and the herds survival rate rising from 89% to 96%; the pig farm that had mixed infection saw the number of piglet death drop from 525 heads to 75 heads, and the herds survival rate rising from 86% to 98%.
The embodiment of the present invention as disclosed above is not meant to limit this invention. All modifications and alterations made by those familiar with the skill without departing from the spirits of the invention and appended claims shall remain within the protected scope and claims of the invention.

## Claims

1. A Mycoplasma vaccine composition for the prevention of swine enzootic pneumonia, comprising two components: a Mycoplasma component comprising 50 to 75% v/v of the vaccine composition and an adjuvant component comprising 25 to 50% v/v of the vaccine composition, wherein the Mycoplasma component comprises 60 to 80% v/v of inactivated Mycoplasma hyopnuemoniae and 20 to 40% of inactivated Mycoplasma hyorhinis ATAIT-7 deposited with American Type Culture Collection under the accession number ATCC PTA-7651.

2. The mycoplasma vaccine according to claim 1, wherein said vaccine is administered to animals via subcutaneous or intramuscular route.

3. A pharmaceutical composition for the prevention of mycoplasma infection, comprising an effective amount of mycoplasma vaccine of claim 1 and a pharmaceutically acceptable carrier.