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Recently, our lab reported that CUBIC, a tissue-clearing technique, could help reveal the initial route of infection, its spread, and the localization of pathogens in infected goldfish Carassius auratus. ​However, this technique, in its original form, failed to clear melanin pigmented parts of the fish. ​We show here that bleaching with H2O2 clears melanin pigments of Black Moor goldfish without causing severe histological damage. ​Furthermore, nuclear staining with PI helps visualize the internal structures of cleared fish in a dark field. ​Since bleaching does not significantly quench the fluorescence of GFP, it may be applied to the in vivo imaging of GFP.

We inoculated yellowtail Seriola quinqueradiata with a commercial bivalent vaccine containing inactivated Vibrio anguillarum and Lactococcus garvieae to better understand the immunological responses to the vaccination. Serum samples were collected from vaccinated and control (PBS-injected) fish at 1, 3, 5, 7, 10, and 14 days post-vaccination (dpv). Compared to the controls, the serum from the vaccinated fish inhibited the proliferation of V. anguillarum from 7 dpv. However, vaccination had no effect on L. garvieae proliferation. The antibody titer against V. anguillarum obviously increased from 7 dpv, but no such increase was detected for L. garvieae. We confirmed that the protection conferred by the V. anguillarum vaccine was a result of the antibodies against the pathogen. However, humoral factors related to protection were not observed for L. garvieae. The expression of immune-related genes in the head kidney was profiled by microarray analysis. Twenty-two immune-related genes were up-regulated during the experimental period. Up-regulation of cellular immune-related genes, such as tumor necrosis factor, CC chemokine, and macrophage colony-stimulating factor receptor, was mostly observed from 1 to 3 dpv, whereas that of humoral immune-related genes was observed from 3 to 14 dpv. Notably, immunoglobulin-related genes showed continuous up-regulation during the 2-week experimental period.

We compared the sensitivity and specificity of PCR methods targeting 16S rDNA, DNA gyrase subunit genes (gyrA, gyrB) and peptidyl-prolyl cis-trans isomerase C gene (ppiC) for the detection of Flavobacterium psychrophilum using 82 bacterial isolates and 55 washings of fish gills. To identify F. psychrophilum among the bacterial isolates, the PCRs targeting 16S rDNA, gyrB and ppiC exhibited the same sensitivity and specificity but some false-positive results were found in the PCR targeting gyrA. The PCR targeting 16S rDNA was more sensitive than the other PCRs in detecting the bacterium from gill washings but occasionally gave false-positives. To avoid the false-positive result in gill washings, the PCRs targeting gyrB and ppiC seem to be preferable.

We cloned and analyzed a complement regulatory protein-like cDNA termed HCRF-2 from a liver cDNA library of the Japanese flounder Paralichthys olivaceus. The cDNA was 3101 bp in length and the predicted translation product of 737 amino acids contains a hydrophobic signal sequence followed by a stretch of 12 short consensus repeats (SCRs). These SCRs were closely related to those of the previously described SBP1 of complement regulatory plasma protein of barred sand bass. SCR domains Nos. 1 to 12 of HCRF-2 resembled domains Nos. 1 to 10 of SBP1. Recently, we reported a complement regulatory protein-like cDNA designated HCRF that contained 7 SCRs. SCR domains Nos. 2 to 7 of HCRF resembled domains Nos. 12 to 17 of SBP1, but SCR domains of HCRF did not resemble those of HCRF-2. In these comparisons, schematic models of HCRF, HCRF-2 and SBP1 clearly showed their close relationship, indicating that these cDNAs originated from the same ancestral gene.

A species-specific plasmid (pZP1) has been isolated from the fish-pathogenic bacterium Pasteurella piscicida. Two DNA fragments of PZP1 (PZP1-1 with 964 bp and PZP1-4 with 477 bp) were cloned from the plasmidpZP1. Two 20-mer primer sets, PZP1-1a and PZP1-1b, and PZP1-4a and PZP1-4b, were constructed according to the nucleotide sequences of fragments PZP1-1 and PZP1-4. A 484-bp DNA fragment was amplified from template DNA from 40 strains ofP. piscicidaby PCR using the PZP1-1 a/1b primer set. The strains were isolated at different times of the year at different places in Japan and the USA. A 321-bp PCR product was amplified from all of the above strains ofP. piscicidaexcept strain ATCC17911 using the PZP1-4a/4b primer set. No such PCR products were obtained from template DNAs ofBeneckea proteolytica, Photobacterium damsela, Ph. histaminum, Ph. leiognathi, 15standard strains ofVibriospp. or four fish pathogenic bacteria (Aeromonas hydrophila, A. salmonicida, Edwardsiella tarda and Enterococcus seriolicida). The PCR products were amplified with the PZP1-1a/1b primer set from DNA from the kidney of yellowtail (Seriola quinqueradiata) infected naturally withP. piscicida, but not from DNA from healthy yellowtail.

In many yellowtail Seriola quinqueradiata farms in the vicinity of Uwajima city, Ehime Prefecture, Japan, a new disease has caused heavy mortalities of yearlings, mainly in summer and autumn. Symptoms included anorexia, lethargy with occasional bursts of swimming and redness in the central nervous system. In a histological survey, the most noticeable sign was severe encephalomyelitis. It is suspected that densely packed parasite-like cells (about 4 μm in diameter) in the spinal cords are the causative agent of this disease.