| Mink is one of important fur animal species. Mink farming is widespread in North America, North Europe, Argentina, Ukraine, China, and so on. With the increase of farming scale and rearing density, high density of mink farming has caused propagation of bacterial diseases, which caused huge economic losses. Hemorrhagic pneumonia, caused by Pseudomonas aeruginosa, has been one of the most costly infectious diseases.Bacteriophages (phages) are bacterial viruses and represent one of the most abundant organisms in the biosphere. They offer several unique advantages over antibiotics, including rapid development from bench to bedside, auto-’dosing’, low input, safely, no residue. Therefore, bacteriophages show great potential as alternatives to antibiotics.In recent years, antibiotic misuse and drug-resistant super-bug have highlighted the need for phages as alternative strategies to combat infectious diseases. At present, U.S. Food and Drug Adnimistration had approved phage-based products to control foodborne pathogens during food process. In this study, we studied effect of a lytic phage specific for Pseudomonas aeruginosa on hemorrhagic pneumonia in mink.1. Five suspected P. aeruginosa strains were isolated from lung samples of hemorrhagic pneumonia mink using cetyl trimethyl ammonium bromide selective medium for P. aeruginosa. The five suspected strains, PA1-1, PA5-1-1, PA5-1-2, PA5-2-1 and PA5-2-2, belonged to the species group of P. aeruginosa identified based on physiological and biochemical indexes and 16S rDNA sequence alignment. The 16S rDNA sequences of PA1-1, PA5-1-1, PA5-1-2, PA5-2-1 and PA5-2-2 were submitted to GenBank database. Their accession numbers were KF977856, KF977857, KF977858, KF977859 and KF977860 respectively. According to the operating procedures and criteria of K-B method recommended by American Clinical and Laboratory Standard Institute, resistance of P. aeruginosa strains to antibiotics was determined. These five strains have higher resistance to the widely used antibiotics, including gentamycin and polymycin, than those strains isolated by other research teams in the previous studies. The level of exotin A released in culture supernatant was detected using the method of ELISA. Exotin A produced by these new P. aeruginosa isolates were higher than did others isolated by other research teams in the previous studies.2. PA0205 and PA5-1-1 were non-lysogenic bacteria, identified using mitomycin C induction. Two phages, PPA0205 and PPA-ABTNL, were isolated from hospital sewage using double-layer agar plates, when PA0205 and PA5-1-1 strains were taken as hosts. Transmission electron microscopy observation showed that PPA0205 and PPA-ABTNL belonged to the family of Siphovirus and Podoviridae, respectively. Host range assay showed that PPA0205 and PPA-ABTNL phages inhibited the growth of 2 and 14 strains among 15 P. aeruginosa isolates tested, respectively, suggesting that PPA-ABTNL phage had broad host range within the species of P. aeruginosa. One-step growth curve analysis of PPA0205 and PPA-ABTNL phages revealed that their latent periods were 30 min and 25 min, respectively and their rise periods were 40 min and 35 min, respectively. The average burst size of PPA0205 and PPA-ABTNL phages was estimated about 70 and 115 PFU per infected cell, respectively. PPA-ABTNL was more stable in pH range of 3.0-9.0 than was PPA0205. PPA-ABTNL also show higher temperature stability in the range of-20-65℃ than did PPA0205. PPA0205 and PPA-ABTNL inhibited growth of their hosts and PPA-ABTNL phage had higher antimicrobial activity than did PPA0205 phage.3. PPA-ABTNL phage was purified by CsCl gradient ultracentrifugation. The genome of PPA-ABTNL phage was isolated, sequenced and analysed. Results of whole genome sequencing and bioinfonnatics analysis showed that the genome comprised 43227 bp (62.4% G+C) and encoded 54 proteins. PPA-ABTNL genome consisted of three regions, which performed the function of the host conversion, DNA replication and coding for structural and lysis proteins, respectively. Of note, RNA polymerase gene was located in the front of genes coding structural and lysis proteins. Comparative genomics showed that PPA-ABTNL had similar genome architecture to phage phiKMV, LKD16 and LUZ19. Genes encoding DNA polymerase, RNA polymerase and major capsid in PPA-ABTNL had high similarities to those of other phages in the genus of phiKMV-like viruses. Holin was predicted to have a transmembrane region among which the structure of 28-50th animo acids was a-helix and the proportion of hydrophobic amino acid was 69.6%. Regulatory elements analyses showed that PPA-ABTNL encoded four host promoters, three phage promoters, and three factor-independent terminators. Data conducted by sodium dodecyl sulfate polyacrylamide gel electrophoresis and mass spectrometry showed that ORF 38 and ORF 42 encoded major capsid protein and injecting needle protein, confirming bioinformatic analysis data. Taken together, these findings indicated that PPA-ABTNL was a member of the genus "phiKMV-like viruses"and did not contain genes encoding ligase, transposase, and toxin proteins. Thus, PPA-ABTNL was a lytic phage and gave it the potential to use in phage therapy.4. Twenty BALB/c mice (weighing 20±2g, half male and half female) was intranasaly administrated with 20μl physiological saline containg 1012 PFU/ml phage PPA-ABTNL or physiological saline for three consecutive days, followed by a four-day recovery period. Slight inflammatory cells infiltration in lung and liver were found in phage-trated mice. Additionally, all mice behaved normally.Thus, it was concluded that administration of a crude preparation ofphage PPA-ABTNL had no significant effect on the health of animals. These results indicated that crude phage preparation was safe to mice administered via inhalation. The titre of phage preparation was not decreased significantly by the atomization by ultrasonic treatment. Therefore, we applied phage PPA-ATBNL by method of ultrasonic spraying to cure mink hemorrhagic pneumonia. Thirty two Jinzhou Black mink (six months) was randomly divided into four groups and anesthetized by intra-peritoneal injection of chloral hydrate followed by intra-nasally challenge with 20μl of physiological saline containing 1 ×108 CFU P. aeruginosa PA5-1-1. Two hours after challenge, all mink in each group were placed into a plexiglass chamber (L×W×H:120 × 50 ×40 cm), and the sprayer was run for 3 min at 3 ml/min to atomize the phages at MOI 1,10,100 or physiological. Then, mink were kept in the chamber for an extra 3 min. The viable number of PA5-1-1 cells (CFU/g tissue) and the titer (PFU/g tissue) of the phages recovered from the lungs was determined 24 h after infection. Results showed that compared to mice in control (non-phage treated) group, the amount of viable P. aeruginosa recoved from lungs in phage-treated mink with the MOI of 1,10 and 100, decreased 5.08%(P>0.05),19.42%(P<0.05), and 26.13% (P<0.05), respectively. The amount of viable phages in phage-treated groups with the MOI of 1,10 or 100, respectively, were 3.33 PFU/g (P>0.05 vs the control group),293 PFU/g (P<0.05 vs the control group) and 13593.67 PFU/g (P<0.05 vs the control group).Through 12 d for phage treatment, the survival rate in phage treated groups in phage-treated groups with the MOI of 1,10 or 100, respectively, were 20%(P>0.05),80%(P<0.05) and 100%(P <0.05), compared to 0% survival rate of the control group. Base on the above analyses, we draw the conclusion that PPA-ABTNL at the dose of MOI 10 can control the mink hemorrhagic pneumonia in the space scale of 30 dm3/mink.Taken together, we obtained two phages, PPA0205 and PPA-ABTNL when P. aeruginosa strains isolated from mink with hemorrhagic pneumonia were taken as hosts and studied their biological characteristics. The genome of phage PPA-ABTNL was analyzed by bioinformatics methods for larger burst size and broader host-range of PPA-ABTNL phage than PPA0205. Our result showed that phage PPA-ABTNL was lytic and belonged to the genus of phiKMV-like. Phage PPA-ABTNL could control mink hemorrhagic pneumonia and increase the survival rate when administered using ultrasonic spraying. These data provide evidence for the phage clinical use and further development of phage PPA-ABTNL in mink hemorrhagic pneumonia and other species with respiratory diseases. |
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