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The PAS-1 possessed approximately 48 kb of ds gDNA which was susceptible to digestion with different restriction endonuclease, and at least 9 structural proteins. To date, 7 phages (phage 25, 31, Aeh1, 44RR2.8t, phiAS4, phiAS5 and phiO18P) infecting Aeromonadaceae have been fully sequenced, and they were classified into Myoviridae in the VIIIth ICTV Report (http://www.ictvdb.org/Ictv/index.htm) as T4-like (phage 25, 31, Aeh1, 44RR2.8t, phiAS4 and phiAS5) and P2-like (phage phiO18P) phages. In general, T4-like and P2-like phages have the common morphology of Myoviridae, whereas the gDNA sizes are considerably different, with approximately 160~250 kb and 31~36 kb, respectively. However, the gDNA size of PAS-1 was quite different from T4-like or P2-like Aeromonas phages, thus indicating that the isolated phage is novel.
Furthermore, our preliminary PAS-1 genome sequencing data revealed that the closest relatives, according to similarity in putative amino acid sequences found in the GenBank database, were the enterobacteria phage phiEcoM-GJ1 and Aeromonas phage phiO18P. The predicted RNA polymerase, DNA polymerase and large subunit terminase protein of PAS-1 were similar to those of phage phiEcoM- GJ1, and the tail fiber and putative muramidase protein were homologous with phage phiO18P. Interestingly, the phage phiEcoM-GJ1 was reported as the first member of a new genus in Myoviridae, which possesses a coliphage T7-like transcriptional system (RNA polymerase) and T1-like DNA packaging system (large subunit terminase) (13). In addition, a potential phylogenetic relationship between Aeromonas and enterobacteria phages was suggested by their genomic (6, 20, 25, 26) and morphological similarity (2). Therefore, it can be assumed that PAS-1 might be genetically similar to phage phiEcoM-GJ1, at least in DNA
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replication, packaging and transcription systems. Moreover, the host range of a phage was determined by its tail fiber genes (35), and PAS-1 showed homology in its tail fiber and muramidase protein with phage phiO18P, which infects A. media (5), thus indicating that it might have similar adsorption and lysis systems against its host cells. Moreover, PAS-1 was also structurally related with other phages;
major capsid protein showed similarity to phage phiEcoM-GJ1, and wac fibritin neck whiskers and prohead core protein were similar to other T4-like Aeromonas phages, Aeh1 and 44RR2.8t, respectively. Based on the genomic and proteomic analysis of the phage PAS-1, it was novel but closely related with other Myoviridae phages infecting enterobacter or Aeromonas species. Detailed complete genome analysis and identification of the ORFs will be the subject of future research.
The TTSS gene in A. salmonicida subsp. salmonicida, which is responsible for secretion of the ADP-ribosylating toxin AexT, was encoded on a thermolabile plasmid, and the absence of the TTSS gene ascV disabled bacteria to secreting AexT, even though the strain contained the aexT gene (34). Therefore, we screened for the ascV gene in all 17 A. salmonicida strains used in this study, and A.
salmonicida subsp. salmonicida AS05 strain was selected for further experiments.
In the host cell lysis tests using the AS05 strain, the growths of bacteria was apparently inhibited after PAS-1 inoculations. However, the OD600 values at MOI of 0.01 and 1 were increased after 24 h post phage-inoculation, and phage-resistant bacteria were isolated in all the MOI groups. Therefore, it can be assumed that A.
salmonicida subsp. salmonicida has its own phage-resistant mechanisms and this resistance can also be achieved at high MOI values (100 and 10,000). However, bacterial growth was apparently inhibited until 48 h post phage-inoculation at MOI
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of 100 and 10,000; thus, a high MOI value was chosen for the further experiments in this study. The phage-resistant mechanisms of A. salmonicida subsp.
salmonicida against the phage will also be further investigated in the future.
Furthermore, the fate of PAS-1 and immune response against it in rainbow trout were also investigated. The phage in fish kidneys was detected until 200 h pa, showing gradual reductions in its PFUs, regardless of its presences in the aquarium waters of phage-administrated rainbow trout. Therefore, we hypothesized the development of potential neutralizing activity in rainbow trout serum against phage for two reasons. First, despite the presence of PAS-1 in the aquarium water, it was not detected in phage-administrated fish kidneys after 240 h pa; and second, the PFUs of PAS-1 were not increased or maintained in the aquarium water while they decreased in fish kidneys. This data indicates that the excretion from the fish or leakage from injection-mediated puncture were not the main causes of PFU reduction in the fish kidney. Based on these results, the neutralizing activity of rainbow trout serum against PAS-1 was evaluated from 10 days pa, when the phage was not detected in the fish kidneys. As we expected, the significant neutralizing activities against PAS-1 were observed at 10, 15 and 20 days pa and declined later.
According to the previous results of neutralizing activity against phage MS2 in brown trout (Salmo trutta) (21), the primary antibody production was initiated within the first 7 days pa, and the peak of antibody titer were reached at 14 days pa.
Even though we did not investigate the initiation of neutralizing activity in rainbow trout, it can be assumed that rainbow trout can also obtain humoral immunity against administrated phage because of the phage presence in the fish kidney until 200 h pa. This was also one of the reasons why a high MOI value was chosen for
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Based on these preliminary results, the protective effects of PAS-1 were evaluated with an MOI of 10,000 in rainbow trout furunculosis model. Some therapeutic or prophylactic uses of A. salmonicida phages were previously attempted, but the studies faced several difficulties with failures in fish protections (10, 37). Unlike previous reports, the fish in the phage-administrated groups in this study showed significantly improved survival rates and considerably increased mean time to death values as compared with the control groups. Unlike other fish- pathogenic bacteria, A. salmonicida subsp. salmonicida is capable of causing disease in healthy salmonids at very low levels of infection; estimated LD50 was lower than 10 CFU/ml by intra-peritoneal injection (7), and bacterial challenge with the AS05 strain (2.5 × 102 CFU/fish) caused 100% of mortality in rainbow trout within 4 days (this study). Additionally, clinical furunculosis usually occurs in fingerling and juvenile salmonids as per-acute form with high mortality within a significantly shorter time compared to adult fish (38). Moreover, while administering phages orally in salmonids for therapeutic usage, the fish stomach could be a critical barrier of phage delivery due to low pH (pH 2.5~4.0). Therefore, phage administration time after bacterial infection and its administration route should be considered as the most important factors in phage therapy against furunculosis, and such considerations may help to minimize economic losses in worldwide salmonid culture caused by casual as well as antibiotic-resistant A.
salmonicida.