# Patent

## Title
Bacteriophage having bactericidal activity with respect to Actinobacillus pleuropneumoniae

## Abstract

The present invention relates to a bacteriophage having bactericidal activity against Actinobacillus pleuropneumoniae. Bacteriophage PA1Φ can infect Actinobacillus pleuropneumoniae and kill the same bacteria and is characterized by the genome of 34,553 by represented by SEQ ID NO: 1.

## Background
TECHNICAL FIELD
The present invention relates to a bacteriophage having bactericidal activity against Actinobacillus pleuropneumoniae. 
BACKGROUND ART
Porcine pleuropneumonia occurs worldwide and has a high rate of incidence. In Korea, it ranked as the fifth most prevalent porcine bacterial disease in 2009. This disease can affect all age groups and is tended to occur in-between seasons. Pleuropneumonia in pigs is transmitted mainly by airborne route or direct contact with an infected pig. The disease causes considerable economic losses due to increased mortality, growth retardation, and decreased feed efficiency by chronic type, etc. Recently, immune lowering viral diseases in pigs such as porcine Aujesky's disease, porcine reproductive and respiratory syndrome (PRRS), etc. are prevalent and as the scale of farming increases, the disease appears to be increasing in incidence. In addition, porcine pleuropneumonia is a highly contagious disease with sudden onset and short course, leading to mass death in swine. Therefore, there is a dire need to develop a preparation useful in prevention and treatment of porcine pleuropneumonia.
The causative agent of porcine pleuropneumonia is Actinobacillus pleuropneumoniae. The pathogens of this bacterium are known as polysaccharides, lipopolysaccharide, hemolysin, enterotoxin, IgA protease, bacterial outer membrane protein, etc. Sixteen serotypes are reported and twelve have been described. Serotypes 2 and 5 are most commonly isolated in Korea.
There exists a need to develop an appropriate alternative therapeutic agent for pleuropneumonia in pigs.
Recently, a bacteriophage is being magnified as an alternative to treat bacterial diseases. Due to the preference for eco-friendly methods, use of bacteriophage is of a higher interest than ever. A bacteriophage, or “phage”, is a very tiny microorganism that infects bacteria. Bacteriophages kill bacteria by entering bacterial host cells and replicating. When enough phage offspring are produced host cells will be broken open and die. The phage offspring spill out of the cell and diffuse toward new hosts. Due to its very high specificity, the bacteriophage capable of infecting a specific bacterium is very limited in type. Namely, a specific bacteriophage can infect and kill only specific bacteria while affecting no other bacteria.
Bacteriophage was first found by British bacteriologist Twort in 1915 during his research on the phenomenon that micrococcus colony turns opaque somehow. Independently, French bacteriologist d'Hérelle discovered a microbe that decomposes Shigella disentriae in a filtrate of feces of a patient with dysentery and called the microbe a bacteriophage or bacteria-eater. Since then, bacteriophages against Shigella dysenteriae, Salmonella Typhi, and Vibrio cholerae were further identified.
After the discovery of bacteriophages, a great deal of faith was placed in their use due to their special ability to kill bacteria and many researches were conducted. With the advent of penicillin that was found by Flemming in 1941, antibiotics became popular and widely marketed. Hence, the study of bacteriophages was largely abandoned in the West except for some East European countries. However, limitations of conventional antibiotics due to antibiotic resistance that have been reported since 2000 have led to a resurgence of interest in bacteriophage as an alternative to antibiotics.
DISCLOSURE
Technical Problem
The inventors of the present invention confirmed that the isolated bacteriophage had bactericidal activity against Actinobacillus pleuropneumoniae and inhibitory activity against growth of Actinobacillus pleuropneumoniae. Based on the finding above, the present invention was finally completed.
Therefore, it is an objective of the invention to provide a bacteriophage useful in prevention and treatment of Actinobacillus pleuropneumoniae infection.
Technical Solution
The present invention provides a bacteriophage capable of infecting Actinobacillus pleuropneumoniae and killing the same.
The bacteriophage of the present invention is bacteriophage PA1Φ having a double-stranded DNA having a size of 34,553 bp and represented by SEQ ID NO: 1. The whole genome information of bacteriophage PA1Φ, which had been identified by the present inventors, was designated as GenBank Accession Number HM624080. The bacteriophage PA1Φ was deposited at Korean Collection for Type Cultures in the Korea Research Institute of Bioscience and Biotechnology, 111 Gwahangno, Yuseong-gu, Daejeon 305-806, Republic of Korea on Oct. 26, 2010 (Accession No. KCTC 11796BP).
As the bacteriophage PA1Φ of the invention can kill Actinobacillus pleuropneumoniae, it can be used in prophylaxis (prevention of infection) or treatment (management of infection) of various diseases caused by Actinobacillus pleuropneumoniae. 
The term “treatment” used herein refers to (i) inhibition of a disease caused by Actinobacillus pleuropneumoniae; and (ii) alleviation of a disease caused by Actinobacillus pleuropneumoniae. The use of bacteriophage for the treatment of bacterial infections in farm animals has been demonstrated (see, for example, Wall, S. K., et al. (2010) Appl Environ Microbiol. 76(1):48-53; Huff, W. E., et al. (2003) Avian Dis. 47(4):1399-405; Callaway, T. R. (2010) Foodborne Pathog Dis. 8(2):261-6)
As used herein, the term “isolated” refers to material removed from its natural environment in which the material occurs. Isolated material undergoes a manmade transformation from its natural state. Isolated material encompasses isolated bacteriophage PA1Φ or particular bacterial isolates including the bacteriophage PA1Φ, isolated and cultured separately from the environment in which it was located. The isolates can be present in purified compositions that do not contain any significant amount of other bacteriophage or bacterial strains, respectively.
The invention further contemplates “variants” of the bacteriophage PAN), which are bacteriophage having minor variation(s) in the genomic sequence and polypeptides encoded thereby while retaining the same general genotypic and phenotypic characteristics as the bacteriophage PA1Φ. Variants of bacteriophage PA1Φ encompass polymorphic variants. Bacteriophage PA1Φ variants capable of performing the same or equivalent biological functions as bacteriophage PA1Φ are particularly preferred. Variant bacteriophage PA1Φ DNA sequences are capable of hybridizing to the referenced SEQ ID NO.:1 under highly stringent conditions (two single strands will hybridize when incubated in 0.1×SSC (15 mM NaCl, 1.5 mM Na3-citrate-2H2O; pH 7.0) at 65° C., but not at higher temperatures.
The bacteriophage PA1Φ of the invention can be used to improve or maintain animal health by preventing or treating bacterial infection. As a result of use of the bacteriophage PA1Φ, the animal may exhibit better performance as reflected by maintaining or improving one or more of the following characteristics: diet, growth, food conversion, survival, appearance, production (e.g., milk production), and/or work rate.
Advantageous Effects
The bacteriophage PA1Φ can provide specific bactericidal activity against Actinobacillus pleuropneumoniae without affecting other useful normal flora. Accordingly, the use of the bacteriophage PA1Φ causes little side effects. In general, use of chemical materials such as antibiotics, etc., causes harm to other normal flora, thereby resulting in various side effects such as lowered immunity, etc.



DESCRIPTION OF DRAWINGS
FIG. 1 is the result of bacteriophage screening showing a clear zone formed on the area spotted by bacteriophage PA1Φ.
FIG. 2 is the result of the assay of bacteriophage PA1Φ for activity of killing various Actinobacillus pleuropneumoniae strains.
FIG. 3 is the result showing growth inhibition of Actinobacillus pleuropneumoniae when the bacteriophage PA1Φ (2×108 pfu/ml) was added in an amounts of 0, 0.125, 0.25, 0.5, and 1 ml to the tubes (in order from the left).



BEST MODE
Hereinafter, the present invention will be described in more detail with reference to the examples, which are set forth to illustrate, but are not intended to limit the present invention.
Example 1
Screening of Bacteriophages for Ability to Kill Actinobacillus pleuropneumoniae 
In order to select a bacteriophage capable of killing Actinobacillus pleuropneumoniae, about 300 types of bacteriophages which had been previously isolated by the present inventors and available at hand, were tested with regard to activity to kill Actinobacillus pleuropneumoniae. These bacteriophages had been collected from pigsty environments or animal tissues.
The activity to kill Actinobacillus pleuropneumoniae was evaluated by spot testing. The assay was performed as follows: Actinobacillus pleuropneumoniae (ATCC 27089) in a 1:1000 dilution was inoculated on a TSB (tryptic soy broth) medium (casein digest, 17 g/L; soybean digest, 3 g/L; dextrose, 2.5 g/L; NaCl, 5 g/L; dipotassium phosphate, 2.5 g/L) supplemented with 2 μg/ml of NAD (nicotineamide adenine dinucleotide) and then shaking-culture was performed at 37° C. overnight to obtain a culture solution. Three ml of the culture solution (OD600 is 2.0) was spread on a TSA (tryptic soy agar) plate (casein digest, 15 g/L; soybean digest, 5 g/L; NaCl, 5 g/L; agar, 15 g/L) supplemented with 2 μg/ml of NAD and then the plate was allowed stand on a clean bench for about 30 minutes so as to dry the liquid spread on the medium. Ten μl at a time of the previously prepared bacteriophage suspension was dropped onto the plate inoculated with Actinobacillus pleuropneumoniae at a time and the plate was allowed to stand for 30 minutes again so as to be dried.
The bacteriophage suspension used in the present testing was prepared by a conventional method (Korean Patent No. 10-0781669), said method is hereby incorporated by reference. The procedure of the method is explained in brief as follows: the bacteriophage culture solution which had been prepared using an appropriate host bacterium was centrifuged at a speed of 8,000 rpm for 20 minutes so as to harvest a supernatant. The supernatant thus harvested was filtrated with a 0.45 μm filter and PEG and NaCl were added to 100 ml of the filtrate so as to make 10% polyethylene glycol (PEG) 8000/0.5 M NaCl. The mixture thus made was allowed to stand for 2-3 hours at room temperature, followed by centrifugation at a speed of 8,000 rpm for 30 minutes to obtain a bacteriophage precipitate. The resultant precipitate was resuspended in 5 ml of a buffer (10 mM Tris-HCl, 10 mM MgSO4, 0.1% gelatin, pH 7.5). The resultant suspension was used as the bacteriophage suspension.
The bacteriophage suspension was spotted onto the plate, followed by static culture at 37° C. for a day. The area covered by the spot was checked for clearing. Clearing of the spot area presumes the Actinobacillus pleuropneumoniae killing activity of the bacteriophage tested.
Based on the results of the assays conducted on about 300 types of bacteriophages, a bacteriophage could be screened for clearing on the spot, i.e., activity of killing Actinobacillus pleuropneumoniae. The bacteriophage showing activity of killing Actinobacillus pleuropneumoniae was bacteriophage PA1Φ. FIG. 1 shows the clearing on the spot area by bacteriophage PA1Φ, observed during the screening procedures. Bacteriophage PA1Φ has been previously deposited by the present inventors at Korean Collection for Type Cultures in the Korea Research Institute of Bioscience and Biotechnology (Accession No. KCTC 11796BP). The entire sequence information of the bacteriophage PA1Φ was also identified as GenBank Accession Number HM624080 by the present inventors.
Bacteriophage PA1Φ needs a suitable host strain such as Pseudomonas aeruginosa. The results of the present working example confirmed that bacteriophage PA1Φ can infect Actinobacillus pleuropneumoniae and kill the same.
Example 2
Investigation of Bacteriophage PA1Φ Killing Activity Against Actinobacillus pleuropneumoniae 
In order to reconfirm killing activity of bacteriophage PA1Φ against Actinobacillus pleuropneumoniae, standard and isolated strains of Actinobacillus pleuropneumoniae were tested for bacterial lysis by spot testing. The spot testing was performed in the same fashion as described in EXAMPLE 1. The Actinobacillus pleuropneumoniae strains used were ATCC 27089, ATCC 27090, ATCC 33377, BA01849, and BA01850. ATCC 27089, ATCC 27090, and ATCC 33377 are standard strains, and BA01849 and BA01850, which were provided by National Veterinary Research & Quarantine Service, are isolated strains. As shown in FIG. 2 illustrating the result from this assay, bacteriophage PA1Φ had killing activity against various Actinobacillus pleuropneumoniae strains.
Consideration of the foregoing results indicated that bacteriophage PA1Φ can be used for prevention and treatment of Actinobacillus pleuropneumoniae infection.
Example 3
Investigation of Inhibitory Activity of Bacteriophage PA1Φ Against Growth of Actinobacillus pleuropneumoniae 
It was examined whether the presence of bacteriophage PA1Φ can inhibit growth of Actinobacillus pleuropneumoniae. To a TSB medium comprising 2 μg/ml, NAD was inoculated in a 1:1000 ratio and shaking culture was then performed overnight. The culture solution thus prepared was diluted with a TSB medium supplemented with a 2 μg/ml of NAD such that OD600 is adjusted to 1.0. To the dilution was added the bacteriophage PA1Φ suspension (2×108 pfu/ml) in an amount of 0, 0.125, 0.25, 0.5, and 1 ml, respectively, followed by addition of a TSB medium supplemented with 2 ug/ml NAD such that the final volume became 2 ml. The culture solution thus prepared was shaking-cultured at 37° C. overnight. The result of the shaking-culture was shown in FIG. 3. As seen from said result, as the amount of bacteriophage PA1Φ increased, the growth of Actinobacillus pleuropneumoniae was slowed.
The results described above confirmed that bacteriophage PA1Φ can inhibit growth of Actinobacillus pleuropneumoniae, and thus, it can be used for preventing infection by the same bacteria.
While the specific embodiments of the present invention have been described above, it is apparent to those skilled in the art that they are only preferred embodiments and the scope of the invention is not limited thereto. Thus, the scope of the present invention is substantially defined by the claims attached hereto and equivalents thereof.
[Number of Accession]
Name of Accession Institute: Korean Agricultural Culture Collection
Number of Accession: KCTC 11796BP
Date of Accession: Oct. 26, 2010

## Claims

1. A method for treating Actinobacillus pleuropneumoniae infections, comprising administering an isolated bacteriophage PA1Φ deposited under Korean Collection for Type Cultures Accession No. KCTC 11796BP to an animal subject, wherein the said bacteriophage PA1Φ has killing activity against Actinobacillus pleuropneumoniae strains.

2. The method according to claim 1, wherein the bacteriophage PA1Φ has a genome comprising SEQ ID NO: 1.