KR101491793B1 - Novel Actinobacillus pleuropneumoniae serotype 12 strain, diagnostic composition and vaccine composition comprising the same - Google Patents

Abstract

The present invention relates to a novel Actinobacillus pleuropneoumoniae serotype 12 strain KACC 91770P which has an effect of preventing porcine pleuropneumonia; to a composition for diagnosing porcine pleuropneumonia containing the strain; and to a vaccine composition for preventing porcine pleuropneumonia containing the strain.

Description

[0001] The present invention relates to a novel actinobacillus pleuropneumoniae serotype 12 strain, a diagnostic composition and a vaccine composition comprising the same,

The present invention relates to a novel actinobacillus flourneumonia serotype 12 strain, a diagnostic composition and a vaccine composition comprising the same, and more particularly to a novel actinobacillus flouronius strain having a preventive effect against swine pneumonia Monoclonal serotype 12, a composition for diagnosing porcine pleural effusion comprising the strain, and a vaccine composition for preventing porcine swine pneumonia comprising the strain.

Pig pleural pneumonia is caused by Actinobacillus pleuropneumoniae , which generally progresses acutely and causes a sudden death of growers or finishing pigs. Major symptoms include appetite deficiency, dyspnea, vomiting, cyanosis, High fever, bleeding in the nose and mouth, sudden death.

(BossJT, Janson H, Sheehan BJ, Beddek AJ, et al., 2000). This is a disease that causes severe economic damage to the swine industry due to the slow rate of delivery and delays in shipment, Rycroft AN, Kroll JS, Langford PR. Actinobacillus pleuropneumoniae: pathobiology and pathogenesis of infection. Microbes Infect. 2002. 4: 225-235; Sebunya TN, Saunders JR, Osborne AD. Dose response relationship of Haemophilus pleuropneumoniae aerosols in pigs. Comp Med 1983: 47: 54-56).

Actinobacillus flourneumonia, a causative organism of swine pneumonia, produces various pathogenic factors including Apx toxin. Up to now, 15 types of serotypes have been identified worldwide. Since the cross-reactivity between the serotypes is not perfect, Identifying the serotype is very useful for the management of pleural pneumonia in that country (Barbara E. Straw et al., Disease of Swine 9th Edition, 2006. p.

Actinobacillus fluorescensis has a wide distribution of serotypes prevalent among countries. Serotype 2 is the most prevalent in Europe, while in Canada and the United States, pleural pneumonia is most commonly caused by serotypes 1 and 5 . Therefore, in order to effectively diagnose and prevent diseases, the pathogen characteristics of the pathogens are identified, and representative strains suitable for them are selected and utilized in various fields.

In domestic pig farms, the highest number of pleural pneumonia due to 2nd and 5th out of 15 serotypes is present. In addition, regarding the importation of breeder pigs, there is a report on the occurrence of pleural pneumonia due to serotype 1, 3, 4, 6, 7, 10, 12 in Korea recently.

Therefore, in order to minimize the damage of domestic pleural pneumonia, it is urgent to develop a diagnostic fluid and to develop a preventive drug using Serotype 12, which is prevalent in Korea.

DISCLOSURE OF THE INVENTION The inventors of the present invention have continued to carry out studies to solve the problems of the prior art as described above and have succeeded in isolating a novel strain of actinobacillus flourneumonia serotype 12 in domestic pig pleural pneumonia lesions, Developmental pattern, and genetic characteristics of the strains, and thus obtained a novel strain of actinobacillus fluorescenumi serotype 12 having a preventive effect against swine pneumonia.

Accordingly, it is an object of the present invention to provide a novel strain of actinobacillus fluorescenoma serotype 12 useful for diagnosis and vaccination of domestic pig-derived pleural pneumonia.

In order to achieve the above object, the present invention provides KACC 91770P strain 12 of actinobacillus flourneumoniae serotype having a preventive effect against swine pneumonia.

In addition, the present invention provides a composition for diagnosing porcine pleuropneumonia, comprising the above-mentioned strain of Actinobacillus flourneumoniae serotype 12.

In addition, the present invention provides a vaccine composition for preventing swine pneumonia in pigs comprising the Actinobacillus flourneumonia serotype 12 strain.

The Actinobacillus flourneumoniae serotype 12 strain according to the present invention may be used as a diagnostic antigen for screening outdoors infected pigs or in the form of a vaccine containing the same for the development of a preventive drug against swine pneumonia .

In addition, it is possible to develop an effective vaccine suitable for domestic situation, and it is considered that the economic damage caused by livestock diseases in swine farms will be greatly reduced through improvement of immunity effect on pleural pneumonia and prevention of pleural pneumonia.

Furthermore, the development of new products and strengthening competitiveness of domestic vaccine manufacturers are expected to have a positive effect on the national economy, such as foreign currency savings resulting from the replacement of imported vaccines and the effect of exporting domestic vaccines.

1 shows the results of 16s rDNA sequencing of Actinobacillus fluorescensia KACC 91770P which can prevent pig pleural pneumonia.
Figure 2 shows the results of comparative analysis of genetic correlations using the PFGE method for domestic isolates of Actinobacillus fluorescens.
Figure 3 shows the growth curve of Actinobacillus flourneumonia KACC 91770P, which can prevent pig pleural pneumonia.

The present invention relates to an actinobacillus flourneumoniae serotype 12 strain KACC 91770P having a preventive effect against swine pneumonia.

The Actinobacillus floxunion serotype 12 strain of the present invention is characterized by having the 16s rDNA nucleotide sequence of SEQ ID NO: 1.

The Actinobacillus flourneumoniae strain of the present invention was deposited with the Korean Agricultural Culture Collection (KACC) of the Institute of Agricultural Biotechnology on Feb. 26, 2013, and received the accession number KACC 91770P.

The Actinobacillus flourneumoniae serotype 12 strain of the present invention is a serotype which is not used in the conventional Actinobacillus flourneumonia vaccine and diagnostic fluid, and is a novel strain of Actinobacillus flourneumonia, Genetically, it represents a representative characteristic of domestic isolationism.

The strain of the present invention is characterized in that the growth rate is faster than that of a domestic field isolate, and the strain exhibits the maximum absorbance from 12 hours of culture.

In addition, the strain of the present invention is characterized by high disease activity in a 10-fold concentrated solution, which can exhibit a mortality of 60% in a 10-fold concentrated solution.

In the present invention, the strain is characterized by having various antibiotic resistance, wherein the antibiotic is selected from the group consisting of neomycin, oxytetracycline, spectinomycin and tylosin But is not limited thereto.

In the present invention, the animal is not particularly limited, but may be a pig, preferably a pig, from which the pneumococcus can be infected.

In addition, the present invention relates to a composition for diagnosing porcine pleural effusion comprising the above-mentioned strain of Actinobacillus flourneumonia serotype 12.

In the composition for diagnosing pleural effusion of the present invention, the strain may be inactivated and lost pathogenicity.

In the composition for diagnosing pleural effusion of the present invention, the strain may be attenuated.

The present invention relates to a vaccine composition for preventing swine flu pneumonia, which comprises the above-mentioned Actinobacillus flourneumoniae serotype 12 strain.

In the vaccine composition of the present invention, the strain may be inactivated and lost pathogenicity.

In the vaccine composition of the present invention, the strain may be attenuated.

The Actinobacillus flourneumoniae serotype 12 strain according to the present invention can be very useful as a vaccine strain for the specific diagnosis and prevention of domestic pleural pneumonia infection.

The vaccine using Actinobacillus fluorescenumi serotype 12 strain may be mainly inactivated, and the attenuated strain produced using the strain may also be included.

The inactivating method may be carried out according to a conventional method in the art. For example, an inactivating agent (for example, 1% formalin) is added to the culture solution of KACC 91770P to inactivate the active agent (for example, aluminum hydroxide gel) Can be added to produce an inactivated killed bacterial vaccine.

In addition, the attenuated strain may be attenuated by a conventional attenuation method, for example, by using attenuating chemicals, bacteriophage, leukocyte, etc., or by using genetic engineering techniques, Can be used to produce attenuated strains that have lost or attenuated virulence.

In addition, when the KACC 91770P strain according to the present invention is used in an animal pleural fluid pneumonia vaccine such as a pig, etc., the vaccine can be prepared by a conventional method in the art, and the strain is treated with Actinobacillus flourneumoniae It can be used in combination with and mixed with serotype strains and other disease agents.

A subunit vaccine, a vector vaccine, a chimera vaccine, or a DNA vaccine produced by using the gene of the Actinobacillus floxunion serotype 12 strain of the present invention So that its utilization is very high.

The vaccine may include a veterinarily acceptable mediator or excipient conventionally used in the art, and an adjuvant may be further added. The vaccine may further comprise an adjuvant for enhancing immunity.

When the KACC 91770P strain according to the present invention is used for the diagnosis of pleural pneumonia, it can be produced by a conventional method in the art.

The preparation of the diagnostic solution can be carried out according to a conventional method in the art, for example, by adding an inactivating agent (for example, 1% formalin) to the culture solution of KACC 91770P strain and inactivating it and using it as a diagnostic fluid for flocculation reaction .

In addition, the attenuated strain may be propagated to extract the outer membrane protein and used as an ELISA antigen to produce a diagnostic solution.

Hereinafter, the present invention will be described in more detail by way of examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by these examples.

Example 1 Isolation and Identification of Actinobacillus fluorescens

Actinobacillus flourneumonia KACC 91770P was collected from pig lungs showing typical pleural pneumonia lesions and cultured in a choclet agar anaerobically at 37 ° C for 24 to 48 hours and then obtained a similar pure colonization to obtain biochemical properties The bacteria were identified by inspection. The anaerobic state was formed using Advanced Anoxomat (TM).

Serotype was confirmed by PCR using the Apx toxin gene of Actinobacillus fluorescens. However, because the serotypes 2, 8, and 15 and serotypes 9, 11, 12, and 13 were not correctly distinguished, the species identification and serotype identification were performed by PCR using the specific genes omlA and cps (Rayamajhi et al., J Vet Diagn Invest. 17: 359362, 2005).

Serum types 2, 5, and 6 and serotypes 1, 7, and 12 were correctly identified by PCR using the specific genes omlA and cps (Øystein Angen et al., Veterinary Microbiology. 132: 312318 , 2008). As a result of the serotype identification, it was confirmed that the KACC 71770P strain was Actinobacillus fluorescenoma serotype 12.

DNA fragments of 16S rDNA were PCR-amplified by extracting DNA of KACC 91770P (Genomic cell / tissue spin kit, Genotech).

The primers used in PCR were 2F: 5'-AGAGTTTGATCCTGGCTCAG-3 '(SEQ ID NO: 2), 2R: 5'- ACGGCTACCTTGTTACGACTT-3' (SEQ ID NO: 5 minutes 95 ° C Denaturation 45 sec 55 ° C Annealing 45 sec 72 ° C Extension 60 sec 30 Cycles 72 ° C The last extension was 10 min.

The primers used in the above sequence analysis were 3F: 5'-AGTTTGATCCTGGCTCAG-3 '(SEQ ID NO: 4), 3F': 5'-CCAGCAGCCGCGGTAATACG-3 '(SEQ ID NO: 5), 3R: 5'- GGTTACCTTGTTACGACTT-3' (SEQ ID NO: 6), and the nucleotide sequence was decoded by a cycle sequencing method using ABI BigDye terminator cycle sequencing ready reaction kit v3.1 (ABI).

PCR was carried out at 95 ° C for 2 minutes, 96 ° C for denaturation for 10 seconds, annealing at 50 ° C for 10 seconds, extension at 60 ° C for 3 minutes, It is 30 cycles.

The analyzed nucleotide sequence was compared with the database of NCBI. As a result, the specific nucleotide sequence of Actinobacillus fluorescens was confirmed.

1 shows the result of 16s rDNA sequencing of Actinobacillus fluorescensia KACC 91770P, which can prevent pig pleural pneumonia. As compared with NCBI database, it has 99% homology with Actinobacillus flourneumonia It looked.

≪ Example 2 >

The genotypes of domestic isolates were compared and analyzed by pulsed field gel electrophoresis (PFGE) method, which is widely used as a comparative genotyping method in US CDC for Actinobacillus fluorescensis isolates.

PFGE is the complete chromosomal DNA of the isolate apaI enzyme, and the band was confirmed by electrophoresis on a CHEF mapper apparatus (Bio-rad, Hercules, USA) at 6.0 V / m for 20 to 4 seconds using a ramp pulse.

Each band was analyzed by the unweighted pair group method with arithmetic means (UPGMA) using BioNumerics (V5.10, Applied Maths, Belgium) software.

Figure 2 shows the results of comparative analysis of genetic correlations using the PFGE method for domestic isolates of Actinobacillus fluorescens.

As shown in FIG. 2, when the isolate of actinobacillus flourneumonia serotype 12 isolated from Korea was classified on the basis of 90% relational relationship, there were two genotypes, and the KACC 91770P strain of the present invention was included Were identified as a representative genotype of domestic isolates.

≪ Example 3 > Observation of growth pattern of isolated strains

A variety of domestic isolates (P-APP-9-12-3 and P-APP-11-6-2) and Actinobacillus flourneumonia KACC 91770P strain were inoculated into BHI (Brain Heart Infusion broth) The absorbance was measured at a constant time while culturing.

Actinobacillus flourneumonia KACC 91770P strains were relatively fast compared to various domestic isolates. That is, as shown in FIG. 3 showing the growth curve of Actinobacillus flourneumonia KACC 91770P strain, which can prevent pig pleural pneumonia, KACC 91770P strains showed maximum absorbance from 12 hours of culture.

Example 4: Pathogenicity comparison between test strains

In order to confirm the pathogenicity of KACC 91770P strain, the pathogenicity was compared by intraperitoneal injection in 0.2 ml of 5-week-old mice.

The results are shown in Table 1 below. The KACC 91770P strain of the present invention is characterized by high disease activity in a 10-fold concentrated solution. The strain was killed at 60% in a 10-fold concentrated solution, while the strain P-APP- 9-12-3 were able to observe high pathogenicity differences in all 10 animals.

Pathogenic comparative investigation of KACC 91770P Test strain Inoculation OD
(405 nm) Mouse pathogenicity by bacterial concentration 100 times concentrated liquid 10 times concentrated liquid Undiluted solution 100 times diluted solution KACC 91770P 0.50 0/10 ^a 4/10 10/10 10/10 P-APP-9-12-3 0.49 2/10 10/10 10/10 10/10

^a Survival rate / Inoculation rate

Example 5: Investigation of antibiotic susceptibility pattern

The antimicrobial susceptibility of KACC 91770P strain was evaluated by using 17 antibiotics to determine the minimal inhibitory concentration (MIC).

The results of the measurement are shown in Table 2 below. Of the 17 antibiotics, the antibiotics are resistant to four antibiotics (neomycin, oxytetracycline, spectinomycin, tylosin).

Measurement of antibiotic minimum inhibitory concentration against KACC 91770P strain of the present invention Test antibiotic Experimental range (ug / ml) Minimum inhibitory concentration (ug / ml) Ampicillin 0.25-16 2 Ceftiofur 0.25-8 ≤ 0.25 Chlortetracycline 0.5-8 2 Clindamycin 0.25-16 2 Danofloxacin 0.12-1 ≤0.12 Enrofloxacin 0.12-5 ≤0.12 Florfenicol 0.25-8 ≤ 0.25 Gentamicin 1-16 8 Neomycin 4-32 > 32 Oxytetracycline 0.5-8 > 8 Penicillin 0.12-8 2 Spectinomycin 8-64 > 64 Tiamulin 0.5-32 16 Tilmicosin 4-64 16 Trimethoprim / sulphamethoxazole 2-38 ≤2 Tulathromycin 1-64 > 64 Tylosin 0.5-32 16

According to the present invention, it is possible to develop an effective vaccine suitable for the domestic situation by utilizing the serotype 12 having a high pathogenicity, and to improve the immunity effect on pleural pneumonia and to prevent the pleural pneumonia by preventing the economic damage caused by livestock diseases Respectively.

In addition, the development of new products and strengthening competitiveness of domestic vaccine manufacturers are expected to have a positive effect on the national economy, such as foreign currency savings resulting from the replacement of import vaccines and the effect of exporting domestic vaccines.

National Academy of Agricultural Sciences KACC91770P 20130226

<110> Animal, Plant and Fisheries Quarantine and Inspection Agency <120> Novel Actinobacillus pleuropneumoniae serotype 12 strain,          diagnostic composition and vaccine composition comprising the          same <160> 6 <170> Kopatentin 1.71 <210> 1 <211> 1360 <212> DNA <213> Actinobacillus pleuropneumoniae <220> <221> gene <222> (1) <223> 16s rDNA of Actinobacillus pleuropneumoniae serotype 12 strain <400> 1 gcccttccct cgaacgaaag aaacggctgc tcaccgcctg cccactcatt acgaaccctt 60 agaccgaata cctcccccta ttgatgccct ttgacatcga ttatggcgca ttatagatac 120 ctaatttccc accctgaaag cccggtggac ggtattctac tcgggttcac cctaatccat 180 caaccaatcc atttccgact ggttcggctg ctagagatcg accagactct cctactggtc 240 ggtgtgacct tgactctgtg ccaggtctga ggatgccctc cgtcgtcacc ccttataacg 300 tgttaccccc cttgggacta cgtcggtacg gcgcacttac ttcttccgga agcccaacat 360 ttcaagaaag ccatcgctcc ttccatagtt taaattatct aaaccattaa ctgcaattga 420 tgtcttcttc gtggccgatt gaggcacggt cgtcggcgcc attatgcctc ccacgctcgc 480 aattagcctt attgacccgc atttcccgtg cgtccgccaa ctaattcact ctacactttc 540 ggggcccgaa ttggaccctt aacgtaaagt atgaccagtt gatctcatga aatccctccc 600 catcttaagg tgcacatcgc cactttacgc atctctacac ctccttatgg cttccgcttc 660 cgtcggggaa cccttacatg actgcgagta cacgctttcg cacccctcgt ttgtcctaat 720 ctatgggacc atcaggtgcg acatttgcga cagctaaacc cctaacctga cactcagacc 780 acgggcttcg attgcactat ttagctggcg gacccctcat gccggcgttc caattttgag 840 tttacttaac tgcccccggg cgtgttcgcc acctcgtaca ccaaattaag ctacgttgcg 900 cttcttggaa tggatgagaa ctgtaggtac cttagaacat ctctatgctc tcacggaagc 960 ccttggtact ctgtccacga cgtaccgaca gcagtcgagc acaacacttt acaacccaat 1020 tcagggcgtt gctcgcgttg ggaataggaa acaacggtcg ctaatccagc ccttgagttt 1080 cctctgacgg ccactatttg gcctccttcc acccctactg cagttcagta gtaccgggaa 1140 tgctcatccc gatgtgtgca cgatgttacc gcatatgtct cccttcgttc taccgctgta 1200 cctcgtttag agtgtttcat gcagattcag gcctaacctc agacgttgag ctgaggtact 1260 tcagccttag cgatcattag cgtttagtct tacaacgcca cttatgcaag ggcccggaac 1320 atgtgtggcg ggcagtgtgg taccctcacc caacatggtc 1360 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 2 agagtttgat cctggctcag 20 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 3 acggctacct tgttacgact t 21 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 4 agtttgatcc tggctcag 18 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 5 ccagcagccg cggtaatacg 20 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 6 ggttaccttg ttacgactt 19

Claims (15)

Actinobacillus pleuropneumoniae KVCC-BA1200404 serotype 12 strain (KACC 91770P), isolated from pigs and causing pleural pneumonia in pigs,
The strain exhibited mouse mortality of 100% in a 10-fold concentrated liquid,
The strain has a higher rate of growth than Actinobacillus pleuropneumoniae P-APP-9-12-3, which is a domestic isolate,
The strain is resistant to one or more antibiotics selected from the group consisting of spectinomycin and tylosin. Actinobacillus pleuropneumoniae KVCC-BA1200404 Serotype 12 strain (KACC 91770P). The strain according to claim 1, wherein the strain has the 16s rDNA nucleotide sequence of SEQ ID NO: 1. The strain according to claim 1, wherein the strain has a maximum absorbance at a wavelength of 405 nm from 12 hours of culture. delete delete delete delete delete delete delete delete delete delete delete delete

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