JPS6119607B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a prophylactic and therapeutic agent for preventing and treating mink pseudomonas disease. Hemorrhagic pneumonia caused by Pseudomonas aeruginosa in mink is a major problem not only in Japan but also in many rural countries around the world. That is, once this hemorrhagic pneumonia occurs, the mortality rate is extremely high, and it has become a major problem in terms of the production economy. Since thousands to tens of thousands of mink are kept in breeding farms, treatment with antibiotics is practically impossible. This is because antibiotic treatment must be injected every 2-3 days, which is not only impracticable per se, but the cost of such frequent injections cannot be applied to economic animals. It is something. Therefore, when Pseudomonas hemorrhagic pneumonia occurs in mink, a bacterial strain cultured from dead mink has been cultured to create a killed formol vaccine, and this has been injected into surviving mink for protection. Currently, there are more than 13 types of Pseudomonas aeruginosa based on serotypes, so it is not possible to create a preventive or therapeutic vaccine using a specific strain in advance. This is because killed formol vaccines can, in principle, only protect against infection by strains belonging to the same serotype. The present inventors isolated a serologically common antigen (OEP) from Pseudomonas aeruginosa and demonstrated that it is present in strains belonging to all serotypes.
At the same time, mice immunized with this OEP antigen
It has been proven that it can withstand infection by all of Dr. Fisher's seven Immunotypes. Therefore, this
OEP was used for the prevention and treatment of Pseudomonas in mink with remarkable effects (Patent Application No. 29104 of 1975, Patent Publication No. 2971).
106714). Infection experiments on mink using the OEP vaccine revealed that immunized animals could survive infection with about 100 times the amount of bacteria compared to untreated animals. However, only about 10,000 animals survived the natural infection.
The creation of a better vaccine has been long awaited, considering that it can withstand nearly twice the amount of live bacteria infection. The present invention was made to meet this long-awaited demand, and the gist of the present invention is to provide common anti-infection protection (OEP) obtained from Pseudomonas aeruginosa.
This is a preventive and therapeutic agent for mink Pseudomonas disease, which consists of a two-component combination vaccine containing two of the following: Pseudomonas aeruginosa elastase toxoid and Pseudomonas aeruginosa protease toxoid. The method for producing the above-mentioned two-component vaccine for preventing and treating mink pseudomonas disease according to the present invention will be described. The production and physical and chemical properties of OEP used in the present invention are described in JP-A No. 48-40925 and
Homma, JYand Abe, C, Japan.J.Exp.Med.
, 42, 23-34 (1972), and JYHomma,
Pseudomonasa aeruginosa Infection and its
Serodiagnosis Microbial Drug Resistance，
University of Tokyo Press, 1975, P.267~
279, as well as Chiyoharu Abe, Kenichi Tanamoto, Takeshi Honma, Kazuo Tarutani, Akio Hoshi, 22nd Toxin Symposium Lecture Collection,
Described on pages 45-50 (1975). The method for producing elastase used in the production of the elastase toxoid used in the present invention and its properties are described in Japanese Patent Publication No. 40-27315 and K.
Morihara et al, J.Biol.Chem., 240P.3295~
3304 (1965) and K. Morihara, J. Bacteriol.
88P.745-757 (1964), and K.Bacteriol,
88P.745-757 (1964), and K.Morihara,
Arch.Biochem.Biophys., 123, P.572-588
(1968). When elastase is toxoidized, elastase may be treated with formalin or oxomethanesulfinic acid as in the production of toxoids of general protein toxins. The method for producing the protease used in the production of the protease toxoid used in the present invention and its properties are described in Japanese Patent Publication No. 40-27315 and K.
Morihara et al, Biochem, Biophs, Acta, 73,
P.113-124, P.125-131 (1963) and K.
Morihara et al, Biophys. Acta, 92, P.351~
360, P.361-366 (1964), and K.Morihara,
Arch.Biochem.Biophys., 114, P.158-165
(1966) and K. Morihara, Biochem,
Biophys, Acta, 309, P.414-429 (1973), and K. Morihara, Agr, Biol.Chem., 38, (3),
Described on pages 621-626 (1974). When toxoidizing a protease, the protease may be treated with formalin or oxymethaneselfuate in the presence of an amino acid (eg, lysine). Example (1) Regarding the preparation of a two-component vaccine 100mg of protease toxoid was added to 24.8ml of phosphate buffered saline (PBS).
buffered saline) (M/15, PH7.4) and add 2.5 ml of 10% potassium alum [K ₂ A
₂ (SO ₄ ) ₄・24H ₂ O]. Another 20%
Add 25 ml of Na ₂ HPO ₄ .12H ₂ O to bring the pH to 6.5 and cause complete precipitation. Finally, add 0.3 ml of 1% Marzonin for livestock protection. (1mg protease toxoid/0.3ml) Elastase toxoid is also treated in the same way. Dissolve 100mg of OEP in 5ml of 0.01N-NaOH,
Add 28ml of PBS to this. Then add 3.3 to this solution
Add ml of 10% potassium alum and add 3.3ml of 20
% _{Na2HPO4 was} added. Finally, 0.4 ml of 1% Marzonin was added. (1mgOEP/0.4ml) Immediately before use, mix any two of protease toxoid-potassium solution, elastase toxoid-potassium solution, and OEP-potassium solution for preventive treatment of the two-part vaccine of the present invention. obtained the drug. This mixture 0.5
500μg protease toxoid in ml, 500μg
It contains either 500 μg of elastase toxoid or 500 μg of OEP. The harmful effects of elastase and protease on living organisms have been thoroughly studied, and in actual animal experiments, protease and elastase have been shown to cause skin necrosis and corneal ulcers in small amounts, as well as strong pathological changes such as bleeding in internal organs. I know what to do. Furthermore, protease- and elastase-producing bacteria can cause ulcers on the cornea of mice, but non-protease-producing bacteria cannot cause corneal ulcers. The present inventors have developed a prophylactic and therapeutic agent consisting of a vaccine (dual combination vaccine) containing any two of protease toxoid, elastase toxoid, and OEP, a common antigen for protection against infection. We will describe experimental infections using this two-part vaccine. Experimental method Animals: Male mink (Saphia species) aged 5 to 6 months Vaccine administration method: Subcutaneous or intramuscular injection Challenging test: Pseudomonas aeruginosa No. 5 strain was used. Infection with live bacteria was performed by injecting 0.5 ml of bacterial solution into the nasal cavity through a vinyl tube under ether anesthesia. Measurement of HA: Measurement of protease HA titer and elastase HA titer was carried out by JYHomma, Japan.
It was carried out according to the method described in J. Mxp, Med, 45, 361-365 (1975). OEP-HA titer measurement is done by JY Homma.Japan
It was carried out according to the method described in J. Exp. Med., 43, 185-189 (1973). The immunization method is shown in Table 1.

【table】

[Table] Effect of infection prevention When immunized mink were infected with live bacteria
The HA titer and preventive and therapeutic effects are shown in Table 2. Table 2
As shown in Fig. 3, the LD ₅₀ was 10 ³ to 10 ⁴ viable bacteria in the untreated mink and in the control injected with only potash alum. In contrast, the LD ₅₀ of mink immunized with PT, ET, or PT+ET was 10 ₈ to 10 ⁹ viable bacteria, indicating a significant infection-preventing effect.

【table】

[Table] Next, field examples of the two-component vaccine will be described. This two-part vaccine was used to treat mink hemorrhagic pneumonia caused by Pseudomonas aeruginosa in a certain area of Hokkaido.
This vaccine was significantly more effective than the OEP vaccine alone. Hemorrhagic pneumonia caused by Pseudomonas aeruginosa broke out in mink pigeons somewhere in Hokkaido, and it lasted from the 5th of one month to the 25th of the following month. Mink farms are A (3394 animals), B (2230 animals),
C (2,090 animals), located in three locations close to each other at intervals of 2 to 3 meters. C vaccinated about half of the mink twice in August, but did not vaccinate A and B. C was A because there was almost no damage.
and talks about B. In A, male and female baby mink (5-6 months old) and adults (17-29 months old) were reared at a ratio of 39.5%, 40.5%, and 20%. For B, the rates were 41.8%, 43.8%, and 14.4%. In A, all mink were kept in seven separate barns before natural breeding occurred, as shown in Figure 1.
In B, the animals were housed in two livestock barns when the outbreak occurred, but after the outbreak, they were divided into four livestock barns. On the 5th of a certain month, hemorrhagic pneumonia occurred at A's breeding farm.
Two or three mink died each day during the first 10 days of the 35-day period. After that, the number of deaths rapidly increased, reaching a peak of 133 deaths on the 18th. After that, it decreased rapidly and ended on the 35th day. Total of 3394 heads
914 (26.9%) animals died. Vaccine available 12 days after outbreak
It was implemented from day one. At farm B, a case of hemorrhagic pneumonia death occurred in the non-vaccinated group on the 20th of that month and continued for 34 days. The maximum number of deaths per day did not exceed 11 during the entire period. 123 out of 2230 animals (5.5%) died.
The first vaccination was administered 3 days before the outbreak.
(In cases of hemorrhagic pneumonia, the patient initially appears tired and has respiratory distress. The nose becomes dry and fluid comes out from the nostrils. Breathing becomes increasingly difficult and wheezing occurs. If this happens, death occurs within 24 hours.) ) The strain isolated from the dead mink was Pseudomonas aeruginosa, Type 3. OEP was dissolved in dilute alkali, diluted with M/15 phosphate buffer (PH7.0), and 10% KAI (SO ₄ ) ₂ 12H ₂ O was added as an adjuvant. Protease toxoid and elastase toxoid were also prepared in the same manner. These two types were mixed to form a vaccine (in the case of OEP alone, it was used without mixing). Two intramuscular injections were given 10 days apart. The amount of vaccine and vaccination plan are shown in Table 3.

【table】

[Table] Results of vaccination Antibodies against OEP, protease toxoid, and elastase toxoid were measured by HA reaction. As a result, there was almost no difference in OEP-HA titer between the combined vaccine and the OEP single vaccine. An example of this is shown in FIG. Protease toxoid was also 32 to 512 times more positive approximately two weeks after the final injection. Furthermore, the HA value of elastase antibody was not as high as that of protease HA, but it was 16 to 64 times more positive. All of these antibodies specifically neutralized protease or elastase activity. Looking at the mortality rate of male and female mink in the non-vaccinated group, the mortality rate for males was twice that of females in farm A.
In breeding farms, it was five times more expensive. The mortality rate of parent mink was clearly lower than that of offspring. (See Table 4)

[Table] Table 5 shows the effects of the OEP single vaccine and the combined vaccine containing OEP and the above two toxoids. Mortality rates differ depending on sex and age, so the effect was evaluated for females only, for males only, and for parent mink only for parents. The results are shown in Table 5.

[Table] The combination vaccine containing OEP and toxoid was the most effective (P<0.001) in all livestock houses. A
Looking at the male mink offspring in the 5th barn, natural outbreaks had already occurred here before the vaccination was administered.
The effectiveness of the combination vaccine has decreased slightly (P<
0.01). However, it is worth noting that the effectiveness of the combined vaccine is clear, even though the vaccine was administered in A's 5 barns 12 days after the outbreak and 3 days after the outbreak in 6 barns. In this No. 5 barn
OEP vaccine alone was not effective. Examples of prevention and treatment using the two-component vaccine of the present invention will be described with reference to Table 3 above. In the table, only 3 out of 165 animals died when using the triple vaccine in the category M in farm B. Furthermore, these three animals died immediately after the injection, so they are not included in the numbers used to determine the effectiveness of the vaccine. In other words, there are actually 0 deaths. As mentioned at the end of the note, out of 165
59 dogs were vaccinated with a combination vaccine of OEP and protease toxoid. The remaining 106 animals received the triple vaccine. That is, in this case, elastase toxoid has almost no meaning. It is thought that only two types were sufficiently effective. Depending on the infecting bacteria, if the number of elastase-producing bacteria is large and protease production is absent or weak, it is considered that OEP and elastase are effective. Next, there is no significant difference in the effect of OEP in the term M of barn 5 of farm A compared to the control without treatment. but
There is a significant difference in OEP immunized with two toxoids compared to the control group, and at the same time there is a significant difference compared to OEP alone. This fact indicates that immunization with a combination vaccine of two types of toxoids, protease and elastase, was effective in treatment, considering that immunization with OEP alone had little efficacy in natural outbreaks.

[Brief explanation of the drawing]

Figure 1 is a diagram showing the configuration of a mink breeding farm in which the preventive and therapeutic agent according to the present invention was administered, and Figure 2 is a diagram showing the mink breeding facility with and without the preventive and therapeutic agent according to the present invention.
1 is a graph of an example showing HA reaction.

Claims (1)

[Claims] 1. A two-component vaccine for mink P. aeruginosa infection consisting of a common protective antigen (DEP) obtained from P. aeruginosa and two of P. aeruginosa elastase toxoid and P. aeruginosa protease toxoid. Preventive treatment agent.