| Vibrio parahaemolyticus is one of the most important zoonotic pathogen, which usually causes vibriosis in marine animals, such as fish, shrimp, shellfish, and crabs, but also infections even death in human in the summer and autumn of every year. It caused the outbreak of pathogenic bacteria of Penaues.vannamei early pancreatic necrosis liver disease and had a huge annual loss since 2009. Because of resistance problems in aquaculture process, antibiotic has been ineffective, so it become very urgent to expand and develop of new probiotics. Phages are bacterial viruses with host specific and high abundance in nature, there are no damage to the natural environment, and no resistance effect and no residual which antibiotics have. Because the phage will die step by step following with the host bacteria gradually death, but also with the proliferation of the host bacteria proliferate themselves. It is durable and strong after phage infect host bacteria, and its usage amount just need a little when lysis happend that will release a lot of phage. Using phage will not cause harm to humans and other aquatic animal, according to the advantage, the use of natural enemies of the bacteria to solve the problem of drug resistance is a novel biological control path.This study choose the V. parahaemolyticus( Vp13) which isolated from P. vannamei, as the host bacteria, to isolate the phage of V. parahaemolyticus. Two phages are isolated which are named SX-2 and SX-F. Through the study of experimental and biological characteristics of SX-2 and SX-F carried out, as well as the protective effect of two phage s with infection V. parahaemolyticus Vp13 to zebrafish. The main contents and results of this study are as follows:1. Isolation and characterization of Vp13 lytic phage SX-2 and SX-F from Vibrio sp.From farmed vannamei ponds and East Coast waters and marine products wholesale market, a total of 77 water samples were collected and enriched V. parahaemolyticus phage against Vp13 overnighet and screened through 0.22 um membrane filter-sterilized. The Vp13 and water samples were mixed while adding Ca Cl2 and PEG and SM buffer, the lytic phage was isolated by double-layer platemethod after 6-10 h. As a result, two phages are isolated which are named SX-2 and SX-F. After pured 5 times, their morphology character were observed by transmission electron microscope Phage SX-2 with a head of about 110 nm and a tail size of about 150 nm. Phage SX-F has an icosahedral head with 56.86 nm in diameter and does not have a tail. Use of bacterial genome extraction kit for phage nucleic acid extraction, At the same time, the DNase1, RNase A and Mung Bean Nuclease and Hind III enzyme digestion assay to identify the type of two phages’ nucleic acid. The results show that: the nucleic acid DNA of SX-2 and SX-F can be digested by DNase I and cannot be digested by RNase A, indicating that the genetic material is DNA; and they both cannot be digested by Mung Bean Nuclease, but can be digested by Hind III, indicating that their nucleic acid are both double stranded DNA; and only a belt. As a result, SX-2 and SX-F nucleic acid are both linear double stranded DNA bacteriophage. According to the ninth report of the classification criteria of the International Classification Committee of the virus, SX-2 belongs to the family Myoviridae; while the SX-F belongs to the family Tectivirus.2. Characteristics of bacteriophage biology and protection against infection Vp13 zebrafishTemperature, p H, etc have a significant impact with phage activity, this experiment showed SX-2 and SX-F have a good temperature resistance, 60 ℃ can survive, but temperatures above 70 ℃ and handling 1h, phage SX-2 and SX-F would be inactivated, but at 60 ℃, treatment 1h, SX-2 SX-F and can survive. SX-2 and SXF are sensitive to p H, when the p H is more than 3, and phage SX-2 SX-F will be deactivated when the p H is greater than 11, and phage SX-2 SX-F will be deactivated. Moreover 30% glycerol stored SX-2 and SX-F at-40 ℃ and-80 ℃ condition was significantly higher than-20 ℃, 4 ℃ and 37 ℃(P <0.05). Because its host specificity, so most of the fragmentation pattern of the phage are very narrow. In this study of 85 test results showed that Vibrio phage SX-2 can lytic 24 kinds of Vibrio.sp which is Vp1, Vp5, Vp3, Vp6, Vp18, Vp21, Vp22, Vp28, Vp33, Vp34, Vp38, Vp44, Vp46, Vp51, Vp52, Vp53, Vp62, Vp79, Vp80, Vp81, Vp82, Vp83, Vp84, K, etc. SXF can lytic 20 kinds of Vibrio.sp which is Vp1, Vp8, Vp9, Vp21, Vp22, Vp25, Vp28, Vp34, Vp38, Vp44, Vp51, Vp52, Vp53, Vp79, Vp80, Vp81, Vp82, Vp83, Vp84, K, etc. In this study, K is V.alginolyticus, others are V. parahaemolyticus. In this study, the two phages’ best MOI is 0.0001. Description two phages have a strong lytic ability. The incubation period of the phage SX-F is about 10 min, lysis period is about 70 min, the burst size is116.2. By the constrast with SX-F, the SX-2 phage incubation period is less than 10 min, lysis period is about 70 min, and the burst size is 209.3. Biological characteristics of the phage showed two phages are lytic phage. In vitro experiments, the phages have a certain influence on the growth of Vp13, its number of viable bacteria can be significantly reduced by both SX-F and SX-2 in the same growth period(24h OD600:SX-2+Vp13, 1.486;SX-F+Vp13, 1.58; Vp13, 1.801). In vivo, we test the protection of phage SX-F and SX-2 through artificially infecting model organism zebrafish with V. parahaemolyticus Vp13. The results showed that two phages have good protective effect, it can reach 62.5%, and the diferent time of administration, the effect of protection also have significant differences, they can get the best protection 50% in 0-4h.3. Genome sequencing analysis of SX-2 and SX-FThe genome size of phage SX-2 is 208581 bp, with 2 t RNA, GC content of 46.84%, a total of 200 open reading frames. The genome size of phage SX-F is 275838 bp, GC content of 48.80%, a total of 101 open reading frames, no t RNA. By gene annotation analysis, SX-2 have 8 tail proteins and 10 structure and function proteins and 23 DNA replication and modification proteins and 1 transport regulatory protein and 1 endolysin protein, and 16 other functional proteins. Gene annotation has 24 genes are not homologous sequences blasted on ncbi; SX-F have 2 tail proteins and 5 structure and function proteins and 11 DNA replication and modification proteins and 1 transport regulatory protein and 1 endolysin protein, and 3 other functional proteins. Gene annotation has 2 genes are not homologous sequences blasted on ncbi;... |
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