JPS626673A - Preparation of strain for live vaccine of pastdurella multocida, live vaccine and strain thereof - Google Patents

Abstract

PURPOSE:To obtain a strain for live vaccine having stable genetic marker and temperature sensitivity, by treating a strain of Pastdurella multocida having infection preventing antigen by variation induction means. CONSTITUTION:A wild type strain of Pastdurella multocida of serum D type having infection preventing antigen, producing hemorrhagic necrotic toxin, is irradiated with ultraviolet rays, incorporated with a variation induction agent such as nitrosoguanidine, etc., and treated by variation induction means to give a strain for live vaccine having saccharide nonfermenting properties, genetic marker and temperature sensitivity, consisting of variant Pastdurella multocida DZ10 strain.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention provides a method for preventing Pasteurella swine infection.
This article relates to a method for producing a live bacterial vaccine strain of Multocida, a live bacterial vaccine, and its strain.

(Prior art) Pig atrophic rhinitis (hereinafter abbreviated as AR) causes atrophy and deformation of the nasal turbinates, causing epidemic pneumonia and growth retardation, making it a major problem in livestock management. . Recently, it has been demonstrated that in addition to Bordetella bronchisepti strains, hemorrhagic necrotizing toxin-producing Pasteurella multocida strains are involved in the severity of AR (de, TO
nglM, P, et, al,, Eng nt1. Pi
g Yet, Soc, 19800ongreas,
Denmark, Proceedings p.
211; Pederaen, K. B & Bar
fod, K., Nord, Vat.-Med.

19B1.55. p, 513-522. ).

In recent years, a killed Bordetella pronkissebutica vaccine has been used to prevent AR, but it has not been sufficiently effective. It has recently been reported that the Pasteurella multocida toxin vaccine is effective in preventing AR (Do Jonget, al., Eng.
, Pig Yet, Sac, 19820ongre
8g.

Mexlao, Proceedingsp, 1 j9)
.

(Means for Solving the Problems) In view of the above-mentioned current situation, the present inventors have developed an effective and safe live bacterium that can protect against AR by preventing infection with hemorrhagic necrotizing toxin-producing Pasteurella multocida. Various research efforts were made to develop a vaccine. As a result, they discovered a method for producing an effective live vaccine strain of Pasteurella multocida that can protect against AR.
The method is characterized by isolating a mutant strain having genetic markers and temperature sensitivity from the d& (Pasteurella multocida) strain by mutagenic means.

The Pasteurella multocida mutant strain obtained in this way was
It has been found that this strain can be distinguished from field strains, has stable genetic identity and temperature sensitivity for more reliable testing as a vaccine strain, and is effective and safe as a live bacterial vaccine.

(Example) Examples of the present invention will be described in detail below.

According to the present invention, hemorrhagic necrotoxin-producing Pasteurella multocida strains are treated with mutagenic agents such as ultraviolet light, nitrosoguanidine, etc.
We have isolated a large number of mutant strains that have non-fermentability (mannitol, etc.), single or multiple genetic markers (e.g. negative for catalase, etc.), and temperature sensitivity (growth is inhibited at temperatures higher than 30°C). From these, by selecting mutant strains with high safety and immunogenicity through animal tests, a live Pasteurella multocida vaccine strain can be obtained.

In this way, for example, the mutant Pasteurella multocida strain DZ10, which has been deposited with the Kikou Institute, is obtained.

The starting strain used in the method of the present invention is preferably a field strain of Pasteurella multocida that produces hemorrhagic necrotoxin of the serotype, but any Pasteurella multocida strain can be used as long as it has the infection-protective antigen of Pasteurella multocida. There may be.

As the liquid medium and the solid medium, any known medium in which Pasteurella multocida strains can grow can be used.

The mutant strain Pasteurella multocida strain DZ10 described above as a representative example obtained by the present invention is a mutant strain of the field-isolated parent strain Pasteurella multocida strain DZ. The field-isolated parent strain Pasteurella multocida strain DZ was isolated from the nasal secretions of pigs suffering from severe AR at a pig farm and was published in the Verge Manual (
Bergey's Manual of Deter
Minative Bacteriology, 8t
h Edition, p, 570-572゜197
This strain is one of the hemorrhagic necrotic toxin-producing strains identified by 5). Pasteurella multocida D21
0 strain has the following biological characteristics (black table 1) when compared with its field-isolated parent strain DZ strain.

Table 1 (Note 1) Inoculate sucrose glycolysis semi-solid agar medium and culture (
Judgment was made after 1 to 3 days at 50°C.

(Note 2) In liquid medium or solid medium, each temperature (50°C9
Growth was determined after culturing at 34°C (937°C, 41°C).

(Note 3) Sterile filtrate of culture supernatant in liquid medium or Gj its dilution 0.1- to guinea pigs) (250-300g weight)
The patient was given a sarcastic injection and evaluated 48 hours later.

(Note 4) Diluted culture solution 0.5! Mouse R1 (SP
? .

OR, 4 weeks old) Injected intraperitoneally and observed for 5 days.

The present invention will be explained below with more detailed examples.

Example 1 First, a strain of Nogustulella that has a genetic marker for non-fermenting of white sugar
We describe the isolation of Multocida mutants. The strain of Pasteurella multocida, which produces hemorrhagic necrotic toxin and is a serotype isolated from the nasal secretions of pigs exhibiting AR symptoms, is called the DZ strain. /Gusterella multocida strain DZ was cultured in YPO liquid medium (Kumagai et al., ed.: Pig Disease, 2nd edition, p. 465, Kindai Publishing, 1982) and the bacterial solution was cultured (37°C, 18 hours) in physiological saline. After centrifugal washing with solution, the cells were suspended again in physiological saline. An aqueous mutagenic nitrosoguanidine solution was added to this at a concentration of 100 micrograms/m/m, and the mixture was heated at 37°C until the survival rate of the bacteria was approximately 1/100.

Immediately, a diluted solution of this treated bacterial solution was spread on an agar plate medium and cultured at 37°C for 2 days. The grown colonies were collected one by one, dispensed into the holes of a microtiter plate (96 holes), and transplanted into a solidified white sugar glycolysis agar medium.

After culturing at 37°C for 3 days, a large number of white sugar non-fermenting colonies were collected in which the white sugar-prepared agar medium did not turn yellow. After repeated single colony separations, stable white sugar non-fermenting mutants were selected.

Next, this white sugar non-fermenting mutant strain was cultured in liquid (57
C. for 18 hours) was washed with physiological saline and then suspended in physiological saline. Take 511L of this bacterial suspension)
Place it in an IJ dish (9cm diameter) and make sure the survival rate of the bacteria is 100~
The ultraviolet rays were irradiated until the intensity decreased to 1/1000. To stabilize mutations, this treated strain was grown in liquid culture (30°
C) and cell division was performed several times. A diluted solution of this culture was spread on a TPO agar plate medium and cultured at 30°C.

Replica the grown bacterial colony onto two ypo agar plates,
One plate was cultured at 40°C and the other at 50°C (3 days). We collected a large number of colonies that grew at 30°C and showed almost no growth at 40°C. After repeated single colony separations, stable temperature-sensitive mutants were selected. One of the strains was called Pasteurella multocida.
multocida) DZ 10 strain (Kikoken Bacterial Report No. 8226). The relationship between the growth (vertical) of the field-isolated parent strain Pasteurella multocida strain DZ and the mutant strain of the present invention Pasteurella multocida strain D210 in a liquid medium at each culture temperature (vertical) and each temperature and 18 hours is shown in the drawing.

As is clear from the figure, the mutant strain of the present invention is found to have temperature sensitivity.

Next, the hemorrhagic necrotic toxin-producing ability of Pasteurella multocida strain DZ10 of the present invention was compared with that of its parent strain Pasteurella multocida DZ strain. The bacterial strain was cultured in liquid (30°C), and 0.1 rnl of the sterile filtrate (0.45 nm Millipore filter) of the culture supernatant was injected into guinea pigs (SPIF,
Hartley, 250-300 g body weight, female) was injected subcutaneously and evaluated 48 hours later. Pasteurella multocida DZ
Injection of the culture filtrate of the strain resulted in the formation of hemorrhagic necrosis with a diameter of 15 to 25 m, and the guinea pig died after 3 days.

However, when the culture filtrate of Pasteurella multocida strain DZ10 was injected, almost no hemorrhagic necrosis was observed, and no death of the guinea pigs was observed (observation for 2 weeks).

Lethal toxicity test: Pasteurella multocida DZ obtained according to Example 1
The lethal toxicity of the 10 strains to mice was compared with their parent strain Pasteurella multocida strain DZ. Bacterial colonies cultured on YPO agar medium (30°C) were suspended in physiological saline. 0.5 of this suspension was intraperitoneally injected into mice (SP?, OR, super, 4 weeks old). Table 2 shows the results of observing the survival and death over time up to the 5th day. Compared to the parent Pasteurella multocida Dz strain, the obtained Pasteurella multocida DZ
It was confirmed that the 10 strains had significantly reduced lethal toxicity to mice.

Table 2 Example 2 Pasteurella multocida DZ obtained according to Example 1
The infection-preventing ability and safety of the IO strain live bacteria vaccine were confirmed in mice. Mouse (SP?, Engineering OR.

Females (3 weeks old) were divided into a control group (10 animals) and an immunized group (10 animals).

After culturing Pasteurella e multocida DZ10 strain on TPO agar plate medium (30°C, 18 hours), it was suspended in physiological saline and the 0. Osm/(3 x 1011 bacteria per mouse) was inoculated intranasally into the mice of the immunized group. Two weeks later, all mice in the control group and the immunized group were injected into the nasal cavity with a culture solution of field-isolated Pasteurella e multocida DZ strain 0.0.
The mice were inoculated and challenged with 5 trrl (2 x 10" bacteria per mouse). The survival of the mice was observed for 15 days, and the results are shown in Table 3.
The DZ10 strain of Pasteurella multocida has an excellent protective ability against infection with the hemorrhagic necrotoxin-producing Pasteurella multocida strain, and no abnormal clinical symptoms were observed in mice in the immunized group. It was confirmed that the strain is highly safe.

Table 3 Example 3 Under the same conditions as shown in Example 2, a test was conducted on the effectiveness of the Pasteurella multocida DZ10 live bacterial vaccine in preventing infection. However, challenge was performed with simultaneous inoculation of Pasteurella multocida strain DZ and Bordetella pronxeptica S strain (2 x 106 bacteria Wl per mouse, respectively).

The results are shown in Figure 4. As described above, the Pasteurella multocida DZ10 strain of the present invention was shown to have a protective effect against simultaneous infection with Pasteurella multocida strain and Bordetella pronchicepti strain, which are the causative strains of AR.

Table 4 Example 4 Pasteurella multocida DZ obtained according to Example 1
A test of the effectiveness of the 10-strain live bacteria vaccine in preventing infection was conducted on pigs. The 9 piglets were divided into 3 groups: 3 animals in the infected control group, 3 animals in the vaccinated infected group, and 3 animals in the non-vaccinated, uninfected control group. A culture solution of Pasteurella multocida strain DZ10 (1.0 x 10' bacteria per pig) was inoculated into the nasal cavity of a 7-day-old piglet. Two weeks after vaccination, a culture solution of Bordetella pronchiseptica S strain (Zhangsha) (1 per pig) was administered.
．． Intranasal challenge with 2 x 10' bacteria) and culture of Pasteurella multocida DZ strain (Zhangsha) one and two weeks later! ! The animals were challenged nasally with a bacterial solution (4.0XIO? bacteria per pig). Thereafter, the head pig was necropsied 7 weeks later (when the pig was 12 weeks old). Severe nasal turbinate atrophy was observed in three dogs in the infected control group. In contrast, only slight nasal turbinate atrophy was observed in the three dogs in the vaccinated/infected group. The nasal turbinates of the three animals in the non-inoculated and non-infected control group were normal. The above results confirmed the effectiveness of Pasteurella multocida DZ10 strain as a live bacterial vaccine.

(Effects of the Invention) As described above, according to the present invention, a mutant strain having a genetic mark and temperature sensitivity was created by mutagenic means from a Pasteurella multocida strain having an infection-protective antigen. provides excellent preventive effects against AR.

[Brief explanation of drawings]

The figure is a graph showing the temperature sensitivity of the strain of the present invention. Cultivated gourd shop (”C)

Claims (1)

[Claims] 1. Pastdurella having an infection-protecting antigen
1. A method for producing a live Pasteurella multocida vaccine strain, which comprises isolating a mutant strain having genetic markers and temperature sensitivity from a Pasteurella multocida strain by mutagenic means. 2. A live Pasteurella multocida vaccine that prevents swine Pasteurella infection using a mutant strain with genetic markers and temperature sensitivity. 3. Pasteurella multocida strain DZ10, a mutant strain having a genetic marker of non-fermenting white sugar and temperature sensitivity (Feikoken Bacterial Serial No. 8226).