Bacteriophage control of Pseudomonas plecoglossicida infection in ayu Plecoglossus altivelis.
Two previously isolated phages were used to examine the therapeutic effects against Pseudomonas plecoglossicida infection in ayu Plecoglossus altivelis. Phage PPp-W4 (Podoviridae) inhibited the in vitro growth of P. plecoglossicida more effectively than Phage PPpW-3 (Myoviridae), and a mixture (PPpW-3/W-4) of the 2 phages exhibited the highest inhibitory activity. In phage therapy experiments, ayu were fed P. plecoglossicida-impregnated feed (10(7) CFU fish(-1)) and then fed phage-impregnated feed (10(7) PFU fish(-1)). Mortalities of fish receiving PPpW-3, PPpW-4, PPpW-3/W-4, and a control fish receiving no phages were 53.3, 40.0, 20.0 and 93.3%, respectively. Phage (PPpW-3/W-4)-receiving fish also showed high protection against water-borne infection with P. plecoglossicida. In a field trial, when phage (PPpW-3/W-4)-impregnated feed was administered to ayu in a pond where the disease occurred naturally, daily mortality of fish decreased at a constant level (5% d(-1)) to one-third after a 2 wk period. The causal relationship of phages in this phenomenon was verified by the long-lasting appearance of administered phages in the kidneys of the fish, and a disappearance of P. plecoglossicida from apparently healthy fish. Neither phage-resistant organisms nor phage-neutralizing antibodies were detected in diseased fish or apparently healthy fish, respectively. These results indicate the potential for phage control of the disease. (+info)
Type II antifreeze protein from a mid-latitude freshwater fish, Japanese smelt (Hypomesus nipponensis).
A lot of reports of antifreeze protein (AFP) from fish have been published, but no report has mentioned of commercialized mid-latitude fresh water fish which producing AFP in its body fluid. We found that the AFP in the body fluid of Japanese smelt (Hypomesus nipponensis) from mid-latitude fresh water was purified and characterized. The N-terminal amino acid sequence of the Japanese smelt AFP was 75.0% identical to Type II AFP from herring. Results of EDTA treatment and ruthenium red staining suggested that the Japanese smelt AFP had at least one Ca2+-binding domain. Interestingly, the antifreeze activity of the Japanese smelt AFP did not completely disappear when Ca2+ ions were removed. The molecular mass of the Japanese smelt AFP was calculated to be 16,756.8 by the TOF-mass analysis. The Open reading flame of the gene coding for the Japanese smelt AFP was 444 bp long and was 85.0% identical with the entire herring AFP gene. The cDNA and amino acid sequence of the Japanese smelt AFP were the same length as those of herring AFP. (+info)
Efficacy of oral vaccine against bacterial coldwater disease in ayu Plecoglossus altivelis.
The development of a practical vaccination method against bacterial coldwater disease (BCWD) in ayu Plecoglossus altivelis and the efficacy of oral administration of formalin-killed cells (FKCs) of Flavobacterium psychrophilum was investigated. The FKC was administrated at a dose of 0.1-0.2 g kg(-1) body weight to juvenile ayu (0.5 g body weight) every day for 2 wk or on 5 days over 2 wk. Experimental immersion challenge at 3 and 7 wk after vaccination showed significantly higher survival rates than the controls. The results show the effectiveness of oral vaccination against BCWD in ayu. (+info)
Clonorchis sinensis metacercarial infection in the pond smelt Hypomesus olidus and the minnow Zacco platypus collected from the Soyang and Daechung Lakes.
The pond smelt Hypomesus olidus and minnow Zacco platypus were collected from the Soyang and Daechung Lakes in January 2003, and their metacercarial infections was examined by the muscle compression and artificial digestion techniques. In the Soyang Lake, 161 metacercariae of Clonorchis sinensis (0.35 per fish) were harvested from 459 pond smelts examined. Also, 13 metacercariae of C. sinensis (0.43 per fish), 1 of Metagonimus sp., 4 of Echinostoma sp., 148 of Centrocestus armatus and 44 unidentified species were collected from 30 minnows. In the Daechung Lake, 369 metacercariae of C. sinensis (3.69 per fish) and 51 unidentified species were recovered from 100 pond smelts. The metacercariae of C. sinensis were fed to experimental rats, in which the adult flukes were identified. The pond smelts and minnows collected from the Soyang and Daechung Lakes were verified to be the second intermediate hosts and the sources of human C. sinensis infection. (+info)
Molecular and sugar-binding heterogeneity of C-type lectins from Osmerus (Spirinchus) lanceolatus eggs.
Two C-type lectins (OLLafs and OLLafl) were isolated from Osmerus (Spirinchus) lanceolatus eggs using asialofetuin-Sepharose column. OLLafs and OLLafl were eluted with 0.2 M sucrose and 0.2 M lactose from the same column, respectively. OLLafl has been estimated to be a heterodimeric protein composed of H- and L-subunit and involved C-type lectin like domain (CTLD). In this study we revealed that OLLafs was a homodimeric protein composed of L-subunit of OLLafl. Although adding EDTA diminished the hemagglutinating activity of OLLafs, the activity of OLLafl was not influenced. Recombinant lectins (rOLLafl-H and -L) and mutant lectins replaced Cys(123, 131 and 136) with Ala (mOLLafl-L(123, 131 and 136)) were established. The activity of mOLLafl-L(136) was comparable to rOLLafl-L, and rOLLafl-H was 15 times lower than rOLLafl-L. On the other hand, the activity of mOLLafl-L(123) and mOLLafl-L(131) were lower than that of rOLLafl-H. Therefore, Cys(136) may not participate in hemagglutinating activity of rOLLafl-L. In contrast, Cys(123) and Cys(131) may partially contribute this activity. Although hemagglutination inhibition profiles of rOLLafl-L, rOLLafl-H and mOLLafl-L(136) were similar, m-OLLafl-L(131)-induced hemagglutination was not inhibited by any sugars tested even at a concentration of 150 mM. Then, Cys(131) may directly contribute to the sugar-binding capacity of OLLafl. Affinities of mOLLafl-L(123) for these sugars were lower than the others. These results suggest that Cys(136) might contribute to the intermolecular disulfide bond in the rOLLafl-L dimer, and that the intramolecular disulfide bond concerning Cys(131) might important for lectin activity. (+info)
Purification, characterization, cDNA cloning, and expression of asialofetuin-binding C-type lectin from eggs of shishamo smelt (Osmerus [Spirinchus] lanceolatus).
A novel C-type lectin (OLABL) was isolated from the eggs of shishamo smelt [Osmerus (Spirinchus) lanceolatus] by affinity chromatography on asialofetuin-Sepharose. OLABL had a molecular mass of 29 kDa on SDS-PAGE under nonreducing conditions and two subunits with masses of 15 kDa (OLABL-H) and 14 kDa (OLABL-L) under reducing conditions. Thus, OLABL is a heterodimeric protein. cDNA sequence analysis revealed that the H- and L-subunits of OLABL were composed of 137 and 136 amino acid residues, respectively, and showed almost identical (95%) sequences, with slight differences in the N-terminal and C-terminal regions. Since each subunit contained only the characteristic motif of C-type lectin-like domain (CTLD), EPN-E-WND, OLABL is a member of group VII of the CTLD-containing protein family. Although OLABL had an EPN sequence that is known as a mannose-specific motif found in the collectin family, OLABL agglutinated rabbit erythrocytes without the addition of Ca(2+) ion, and this activity was inhibited by l-rhamnose and d-galactose derivatives, but not by d-mannose and d-glucose. These results indicate that OLABL has similar characteristics to AJL-2, a calcium-independent lactose specific lectin isolated from Japanese eel skin mucus. Recombinant OLABLs (rHisOLABLs), His-tagged homodimers of the H- and L-subunits, were refolded from inclusion bodies expressed by Escherichia coli. rHisOLABL-L was recovered as a soluble form, but rHisOLABL-H was hardly dissolved in a renaturing buffer. The specific activities of rHisOLABL-L, rHisOLABL-H, and native OLABL were 500, 36, and 20, respectively. These findings suggest that the combination of subunits may affect the solubility and activity of these dimeric form lectins. (+info)
Pathogenesis of experimentally induced bacterial cold water disease in ayu Plecoglossus altivelis.
Ayu Plecoglossus altivelis were experimentally infected with Flavobacterium psychrophilum, which is the causative agent of bacterial cold water disease (CWD). The fish infected by immersion usually died within an hour after they became moribund. The blood volume and haematocrit values of moribund fish were low, while those values of many infected fish that were not moribund were in the range of controls. Most of the affected fish in the immersion-infected groups had ulcerative lesions on their lower jaw. No histological evidence of haemolysis was observed. These results suggest that rapid bleeding occurred through ulcerative lesions, probably causing hypoxia which killed the fish. Ulcerative lesions developed on the dorsal skin when this area had been slightly abraded artificially prior to immersion challenge. Histologically, F. psychrophilum was initially found on the skin that had microscopic injuries, but not on normal skin. The bacterium then entered the dermis and migrated through connective tissues. The lesions subsequently expanded into the underlying musculature through the myosepta, developed necrotic myositis and formed externally open ulcers. Only in later stages of infection did mild lesions develop in the internal organs and the gill, probably caused by the bacterium migrating through blood vessels. This suggests that infection with CWD through the gill or digestive tract is unlikely. Virtually no open lesions were found in ayu challenged by intramuscular injections except at the injection sites. The results suggest that skin injuries are major portals of entry for F. psychrophilum in ayu, and the bacterium has affinity for collagenous connective tissues. (+info)
Crystallization and preliminary X-ray crystallographic analysis of Ca2+-independent and Ca2+-dependent species of the type II antifreeze protein.
Ca2+-independent and Ca2+-dependent species of the type II antifreeze protein (AFP) were both crystallized using the hanging-drop vapour-diffusion method. It appeared that the crystal of the Ca2+-independent species from Brachyosis rostratus belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 43.3, b = 48.4, c = 59.7 A, and diffraction data were collected to 1.34 A resolution. For the Ca2+-dependent type II AFP species from Hypomesus nipponensis, crystallization was carried out for its Ca2+-free and Ca2+-bound states. 1.25 A resolution data were collected from the crystal in the Ca(2+)-free state, which exhibited P3(1)21 (or P3(2)21) symmetry, with unit-cell parameters a = b = 66.0, c = 50.3 A. Data collection could be extended to 1.06 A resolution for the crystal in the Ca2+ -bound state, which appeared to be isomorphous to the crystal in the Ca2+-free state (unit-cell parameters a = b = 66.0, c = 49.8 A). These data will allow us to determine the high-resolution structures of the two species of type II AFP. (+info)