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.
Respiratory burst and phagocytic activities of turbot (Scophthalmus maximus) leucocytes were determined after an in vitro incubation of head kidneys with different doses of yeast glucan and Photobacterium damsela (formerly Vibrio damsela) O-antigen. Although there was an increment of these immune functions with the treatment of glucans and O-antigen compared with controls, they were inhibited at high doses (200 μg/ml). The optimum dose of glucan was 50 μg/ml when it was administered alone, but a concentration of 10 μg/ml was enough to produce the stimulatory effect.
With 1 hour exposure time, the maximum concentration of NaCl which was not toxic to rainbow trout eggs was 25 ppt at 13°C. The present study showed that interval treatment with 25 ppt NaCl for 1 h, twice a week, decreased fungal infection and increased the hatching rate of rainbow trout eggs. Continuous treatments with NaCl at concentrations of 3, 5, 7 ppt apparently increased the hatching rate to almost the same level. Furthermore, in the group treated with 7 ppt NaCl, no fungal infection was detected.
The survival of Aeromonas hydrophila was studied in a variety of waters containing different concentrations of salts. The survival of A. hydrophila for a period of 120 h was found to be optimal at 0.85 and 0.35% NaCl, 1% KC1, 0.2% CaCl2 · 2H2O or 0.4% MgCl2·6H2O, respectively. Among the formulated salt water solutions examined, the best survival was found in that containing 0.60% NaCl, 0.50% KCl, 0.10% CaCl2· 2H2O and 0.20% MgCl2· 6H2O in distilled water (FW2). Virulence of the bacterium starved for 24 h in FW2 and 0.85% NaCl was compared to that of the bacterium cultured in nutrient broth to carp and goldfish by intraperitoneal injection. The median lethal dose obtained by the infection verified that the bacterium starved in both salt solutions had obviously higher virulence than the cultured bacterium.
The nested PCR was used to detect Cytophaga psychrophila carried by juvenile ayu (Plecoglossus altivelis) and eyed eggs of coho salmon (Oncorhynchus kistch). Ayu juveniles were caught in Lake Biwa and frozen shortly after landed. The coho salmon eggs from the USA and Japan were collected on a lot-by-lot basis at Narita airport or hatcheries when the containers arrived. Chelex 100 was used as a medium for extraction of DNA from these samples. Utilizing the PCR, a specific primer pair of PSY1 and PSY2 for C. psychrophila was used to amplify its 16S rDNA segments. An amplification product of the expected size was obtained from 5 of 24 DNA extractions from the kidney of ayu samples in April 1995. These fish had been previously diagnosed as negative for the presence of C. psychrophila by cultivation on TYE agar and IFAT. Five of 11 lots of coho salmon eggs were found to be positive for C. psychrophila by PCR. Four of these five lots had also been diagnosed as positive by cultivation and IFAT. In addition, the sensitivity of the PCR amplification for detecting C. psychrophila in kidney samples of ayu was calculated to be 1.5 CFU per PCR reaction tube.