Immune responses induced by oil-adjuvanted inactivated vaccine against Flavobacterium psychrophilum in ayu Plecoglossus altivelis.

Oil-adjuvant formulated formalin killed cells of Flavobacterium psychrophilum (FKC + Adj) is strongly effective against bacterial cold-water disease (BCWD) in ayu Plecoglossus altivelis. In this study, we aimed to understand mechanisms underlying the strong protection by the vaccine in ayu. Antibody titer of FKC + Adj and formalin-killed cells (FKC) group was significantly higher than those of modified cytophaga broth injected (MCY) group and MCY with the adjuvant (MCY + Adj) group. The highest antibody titer was observed in FKC + Adj group. Granulomatous inflammation without lymphocyte cuff was observed in the spleen and trunk kidney of FKC + Adj and MCY + Adj group, while the size of the granuloma was bigger in FKC + Adj than in MCY + Adj group. Gene expression level for IL-8 was significantly up-regulated in FKC + Adj group at 4 weeks after the vaccination. In contrast, IL-10 gene expression level was significantly suppressed in FKC + Adj at 4 weeks after the vaccination. F. psychrophilum was almost cleared in the spleen and trunk kidney of FKC + Adj group within 2 days after the challenge. Fluorescent immunohistochemistry showed that a lot of bacterial signals were detected in the spleen and trunk kidney of challenged fish in MCY, FKC and MCY + Adj group. However, the fluorescent signal was not detected in the organs of FKC + Adj group after the challenge. These data suggest that F. psychrophilum is immediately cleared in FKC + Adj vaccinated fish and both specific antibody and activation of phagocytes are essential to clear F. psychrophilum in ayu.

Detection and sequence analysis of DNA polymerase and major envelope protein genes in koi herpesviruses derived from Cyprinus carpio in Gunma prefecture, Japan.

Koi herpesvirus (KHV), which is believed to be an emerging virus, causes fatal diseases in carps. Since the 1990s, the presence of KHV has been confirmed in several countries. In Japan, from 2003 to 2004, large outbreaks of KHV infection in farmed carps resulted in the death of numerous fishes. From April to May 2004, we collected 43 dead or dying carps exhibiting typical symptoms of KHV infection in Gunma prefecture. To conduct a molecular epidemiologic study of KHV in our prefecture, we amplified DNA polymerase and the major envelope protein genes of KHV derived from carp gills using newly designed primers. We also performed sequence analysis of both genes of KHV. Sensitivity of our PCR method for amplification of DNA polymerase and the major envelope protein genes of KHV was 3 x 10(2) (100 fg) and 3 x 10(3) (1000 fg) copies of KHV genome, respectively. We detected both DNA polymerase and major envelope protein genes in 37 of 43 carps (86%). No mutation was found in both the genes sequenced from 11 strains, which included two foreign strains and one domestic strain. The results suggested that KHV strains derived from carps in our prefecture were closely related genetically to the other KHV strains.

Identification of gadoid species (Pisces, Gadidae) by PCR-RFLP analysis.

A rapid PCR-RFLP analysis was optimized to identify the presence of 3 closely related gadoid fish species: Alaska pollack Theragra chalcogramma, Pacific cod Gadus macrocephalus and Atlantic cod Gadus morhua in commercial seafood products. Gadoid universal primers were designed for PCR amplification of a 558-bp fragment encoding the mitochondrial cytochrome b gene. Without purification of the PCR products, double digestion with Eco32I and Eco105I restriction enzymes generated reproducible species-specific restriction patterns visualizing 3 fragments (106 bp, 161 bp and 291 bp) in Alaska pollack and 2 fragments (106 bp and 452 bp) in Pacific cod, whereas no cleavage was observed in Atlantic cod. This PCR-RFLP analysis is simple, rapid and reliable, and therefore can be routinely applied to discover fraudulent substitution among 3 economically important gadoid species in commercial seafood products.

Relationship between gyrA mutations and quinolone resistance in Flavobacterium psychrophilum isolates.

Flavobacterium psychrophilum is the causative agent of the fish diseases called bacterial cold-water disease and rainbow trout fry syndrome. It has been reported that some isolates of F. psychrophilum are resistant to quinolones; however, the mechanism of this quinolone resistance has been unexplained. In this study, we examined the quinolone susceptibility patterns of 27 F. psychrophilum strains isolated in Japan and the United States. Out of 27 isolates, 14 were resistant to both nalidixic acid (NA) and oxolinic acid (OXA), and the others were susceptible to NA and OXA. When amino acid sequences deduced from gyrA nucleotide sequences of all isolates tested were analyzed, two amino acid substitutions (a threonine residue replaced by an alanine or isoleucine residue in position 83 of GyrA [Escherichia coli numbering] and an aspartic acid residue replaced by a tyrosine residue in position 87) were observed in the 14 quinolone-resistant isolates. These results strongly suggest that, as in other gram-negative bacteria, DNA gyrase is an important target for quinolones in F. psychrophilum.

Genotyping of Flavobacterium psychrophilum using PCR-RFLP analysis.

Genetic variability among 242 strains of Flavobacterium psychrophilum was characterized using polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) analysis. Universal Primers GYR-1 and GYR-1R, which were designed to amplify the gyrase subunit B gene (gyrB), yielded a 1178 bp PCR product encoding gyrB and a 290 bp PCR product of anonymous DNA from all F. psychrophilum strains tested. In the RFLP analysis of the anonymous 290 bp DNA marker, the restriction enzyme HinfI generated 2 cleavage patterns (Genotypes A and B). Genotype A was found only in isolates from ayu (n = 109), while Genotype B was found in isolates from coho salmon (n = 11), ayu (n = 35), rainbow trout (n = 43) and other fishes (n = 44). In the second experiment, Primers PSY-G1F and PSY-G1R specific for F. psychrophilum, were used to amplify gyrB. The specific primer pair amplified the expected size (1017 bp) PCR product from all F. psychrophilum strains. In the RFLP analysis of the gyrB, the restriction enzyme RsaI produced 2 genotypes, R and S. Genotype R was found in isolates from coho salmon (n = 6), ayu (n = 27), rainbow trout (n = 39) and other fishes (n = 4). Genotype S was found in isolates from coho salmon (n = 5), ayu (n = 117), rainbow trout (n = 4) and other fishes (n = 40).

Secondary-site mutation restores the transport defect caused by the transmembrane domain mutation of the xenobiotic transporter MexB in Pseudomonas aeruginosa.

It has been suggested that the MexB subunit of the MexAB-OprM efflux transporter of Pseudomonas aeruginosa exports xenobiotics in an energy-dependent manner. To investigate the role of the transmembrane segments (TMS) of MexB in the transporter activity, we isolated 24 spontaneous mutants showing hypersusceptibility to antibiotics. Among them, three mutations were located at TMS-3, TMS-4, and TMS-10 having amino acid substitution Leu376vPro, Gly397vVal, and Val928vGly, respectively. A secondary mutation, which suppressed the defect caused by the Val928vGly mutation in TMS-10, was found at the 403rd amino acid residue in TMS-4 with a change of glycine to serine, suggesting that TMS-4 and TMS-10 may be in close proximity. This result provided strong support for the recent notion that negatively charged residues in TMS-4 might form a salt-bridge with a positive charge in TMS-10 (Guan, L., and Nakae, T. (2001) J. Bacteriol. 183, 1734-1739). The transporter function impaired by the Gly397vVal mutation in TMS-4 was recovered by the secondary mutation, Gln998vHis, in the loop between TMS-11 and TMS-12, thereby suggesting that TMS-4 and TMS-11 or TMS-12 might also be in close proximity. Thus, it is most likely that TMS-4, TMS-10, and TMS-11 or TMS-12 are packed close three dimensionally.