Two-year-old tiger puffer Takifugu rubripes, persistently infected with the monogenean Heterobothrium okamotoi for longer than one year, were cohabitated in an aquarium with one-year-old tigerpuffer with no previous record of infection (naïve fish) for 70 days. The infection level in the naïve fish rose sharply, and the intensity of infection reached its highest on day 30 (estimated 16, 700 parasites on the gills per fish). In the persistently infected fish, on the other hand, no change was recorded in the infection level, which was much lower than that of the naïve fish. Based on the compositions of parasite developmental stages in the two fish groups, we hypothesize that the protection mechanism found among the persistently infected fish functioned in at least three occasions : firstly when the oncomiracidium settled on the gills, secondly when the parasite developed from a worm without clamps to one with a pair of clamps, and thirdly when it migrated and grew on the branchial cavity wall. The persistently infected fish produced anti-Heterobothrium antibodies, while no antibody was detected in the naïve fish. These results suggest that a persistent infection induces protection against H. okamotoi, but the factors responsible for effective prevention against reinfection remain to be clarified.
Nine strains of fungi in the genus Fusarium were isolated from the lesions with black gills of cultured kuruma prawn Penaeus japonicus in Japan between 2000 and 2003. All strains showed same morphological characters. Two strains selected at random showed pathogenicity to kuruma prawns by intramuscular injection. From the precise morphological features of a representative isolate, it was identified as a member of the Fusarium solani complex. Phylogenetic analyses based on the sequences of its internal transcribed spacer region, including 5.8S ribosomal DNA and a partial 28S ribosomal DNA region, showed that all five strains tested were monophyletic.The present strains and the phytopathogenic Fusarium solani were clearly distinguished by the morphological and phylogenetical characteristics.
In vivo egg-laying by the diclidophorid monogenean Heterobothrium okamotoi was studied in artificially infected tiger puffer Takifugu rubripes. After exposed to oncomiracidia, puffers (0-year-old;n=40) were placed individually into an aquarium with recirculating sea water kept at 20°C. Parasite eggs were collected daily by filtration of drained water from the aquarium. The daily egg output per parasite was calculated by dividing the number of collected eggs by the number of parasites determined at the end of the experiment. The egg-laying, recorded for 19-33 consecutive days, fluctuated widely among individual host fish and/or parasites. The number of laid eggs increased for the first 10 days before reaching a plateau, ranging daily from less than 10 to more than 1000 eggs per parasite, with the mean daily output throughout the observation period being 51.2-362 eggs per parasite. Egg strings laid in a single output were calculated to be 2-157 cm long. The frequency of egg-laying, monitored in a fish with a single parasite, ranged from several outputs in a day to no egg-laying for up to 3 days. Egg hatching was unstable for the first few days, but subsequently the hatching rates increased to 90% or higher, irrespective of the number of eggs laid by parasites at different intensities of infection.
Effects of water temperature on infection of the microsporidian Kabatana takedai were investigated in salmonids under field and experimental conditions. “Cysts” of K. takedai appeared in the heart and the skeletal muscle of wild juvenile masu salmon Oncorhynchus masou from the Chitose River, Hokkaido, during summer in 2003 and 2004, when the river water temperature exceeded 15°C. Following the exposure of naive juvenile sockeye salmon Oncorhynchus nerka to river water (18-19°C) for 3 days, the fish were transferred to separate tanks where the temperatures were set to 11, 13, 15 and 17°C. Prevalence of infection reached more than 70% in the 13, 15 and 17°C groups, but in the 11°C group, it remained less than 30%. However, shifting a part of the 11°C group to 18°C increased the prevalence to 71 % by 23 days after the elevation of the temperature. When juveniles were exposed to 11°C river water and subsequently kept at 9°C for 42 days, no development of K. takedai occurred even after the temperature was elevated to 15°C. These results indicate that temperature manipulation is partially effective as a preventive method of K. takedai infection.
Experimental dual-infections with a non-lethal aquabirnavirus (ABV) and a lethal betanodavirus (redspotted grouper nervous necrosis virus : RGNNV) were carried out in Japanese flounder Paralichthys olivaceus and sevenband grouper Epinephelus septemfasciatus. In the dual-infection group, ABV was intramuscularly (IM) injected into fish seven days before the IM-injection with RGNNV. In the experiments with flounder, a high expression of an Mx gene, a molecular marker for type I interferon (s) (IFN) production, occurred in the head kidneys and brains at Day 7 post-ABV injection. Although no mortality was found not only in the dual-infected group but also in the single infection group with RGNNV (control group), the infective titers of RGNNV in the tissues of the dual-infected group were significantly lower at any sampling times than those in the control group. In the experiments with grouper, the preceding ABV infection resulted in complete protection against RGNNV infection. The infective titers of RGNNV in the tissues were also lower in the dual-infected group than in the control group throughout the experiments, and finally the virus disappeared from the head kidneys and brains of the dual-infected group at Day 14 and Day 56 postinjections, respectively. These results suggest that an ABV-induced IFN (s) effectively suppresses the progression of secondary betanodavirus infection.
We examined the pathogenicity of typical (motile) and atypical (non-motile) Edwardsiella tarda against three marine fish species : yellowtail Seriola quinqueradiata, Japanese flounder Paralichthys olivaceus and red sea bream Pagrus major. Through intraperitneal injection, all provided strains (five typical and two atypical strains) showed pathogenicity to yellowtail and Japanese flounder, while only the atypical strains showed pathogenicity to red sea bream. In immersion challenges using each one selected strain, Japanese flounder was killed only by the typical E. tarda, while red sea bream was killed only by the atypical E. tarda. The results show the apparent difference in the pathogenicity between typical and atypical E. tarda.
The virucidal effects of ultraviolet (UV) irradiation, heat treatment and disinfectants against koi herpesvirus (KHV) were evaluated using KF-1 cells. KHV (KHV-I strain) was completely inactivated by UV irradiation at a dose of 4.0 × 103 μ Ws/cm2 or heating to temperatures above 50°C for 1 min. KHV was completely inactivated by 200mg/L iodophor, 60 mg/L benzalkonium chloride solution or 30% ethyl alcohol for 20 min. When chlorine concentration was measured after mixing with virus supernatant containing medium and fetal bovine serum, 97.5%and 98.5%reduction in infectivity was observed at 0.30 mg/L of chlorine for 30 s and 20 min, respectively. In practice, a tenfold concentration of chlorine (i.e.3 mg/L) will be recommended to inactivate KHV completely.
Appearance of Edwardsiella tarda were monitored in pond water culturing eel Anguilla japonica in Kagoshima prefecture. E. tarda were isolated at almost all sampling times. Serotype and siderophore production of the E. tarda isolates, which are involved with its pathogenesis, were determined. Out of 370 isolates, 194 isolates (52%) were classified into serotype A and 246 isolates (66%) produced siderophore. Ninety-one percent of the isolates that were classified into serotype A produced siderophore;72%of those that produced siderophore were classified into serotype A. There was no cross linkage between serotype and siderophore production in E. tarda.
A mass mortality of cultured rockfish Sebastes schlegeli (138-145 g in body weight) occurred in the spring of 2001. Histopathological examination revealed necrotic lesions, aneurysms, hemorrhage, hyperplasia of epithelial cells and lamellar fusion in the gill tissues. Gramnegative bacilli were observed in the gill lesions. In some fish, necrotic foci due to bacterial invasion were observed in the splenic ellipsoid, hematopoietic tissue of the kidney and myocardium. However, no bacteria were isolated from the liver, kidney, spleen or brain.