Infection rates with white spot syndrome virus (WSSV) in 4 small penaeid shrimp species, Metapenaeopsis acclivis, M.barbata, M. dalei and Trachypenaeus curvirostris collected from the western Seto Inland Sea of Japan, was examined by PCR, and transmission of the virus to juvenile Penaeus japonicus was tried by oral or intramuscular inoculation of infected samples. No shrimp collected from the Aki-Nada and Iyo-Nada regions were shown to carry WSSV, except 2 out of 274 individuals of M. dalei from Iyo-Nada. Infection rates of M. acclivis, M. barbata and T.curvirostris collected from the Suo-Nada region were 29, 40 and 23%, respectively. Oral administration of small pieces of PCR positive shrimp muscle did not kill any challenged shrimp during the test period, but could establish infection with WSSV. Half of the challenged shrimp injected with a filtered homogenate of PCR positive shrimp muscle died with typical signs of WSS within 11 days.
Fate of viral hemorrhagic septicemia virus (VHSV) in tissues of Japanese flounder Paralichthys olivaceus (20.7 g in average body weight) challenged by immersion for 1 h with a flounder isolate (Obama 25, an American genotype) of VHSV at 104.0TCID50/mL was monitored for 7 weeks by virus isolation method with FHM cell line. The mortality of Japanese flounder challenged by the same method in a companion experiment reached 68% at 13°C at week 6 postimmersion challenge (PIC). VHSV was detected in the gills just after the immersion, in the kidney, spleen and skin at day 1 PIC, and in all the tissues tested at day 3 PIC. The infective titers of VHSV higher than 109.0TCID50/g were measured in the heart and blood as well as the foregoing tissues except skin at week 1 or 2 PIC. VHSV was not detected from most tissues at week 4 PIC except from the gills, heart, brain and blood, and at week 6 it was isolated only from the heart and brain. When the virus was not isolated from any tissues at week 7 PIC, the surviving fish were given stresses by 3 different methods, i.e. shaking of the fish container, or abrupt raise (13°C to 18°C) or fall (13°C to 8°C) in water temperature. As a result, VHSV was detected from the heart of some fish after the temperature stresses, indicating that the heart is the most probable hiding site of the host for VHSV.
Species-specific polymerase chain reaction (PCR) targeting the 16S rRNA gene of Nocardia seriolae was developed.The PCR targeted nucleotide #609 to 1038 (Escherichia coli numbering), which gave a 432 bp-length product. This method could detectN. seriolaetype strain (JCM3360) and eight clinical isolates of this species from yellowtailSeriola quinqueradiataand Japanese flounder Paralichthys olivaceus, but not those of other bacterial species including 5 otherNocardiaspp. and 4 yellowtail pathogens. The detection limit of the PCR was 102CFU. Eight diseased yellowtail were employed for detection of the bacterium by the PCR. Positive results were obtained from all fish.
We induced three types of triploid rainbow trout by retention of the second polar body (RSP) and cross fertilization between tetraploids and diploids (4_??_×2_??_and 2_??_×4_??_) and found that hemoglobin concentration of triploid (4_??_×2_??_) was higher than those of the others. No difference was observed in their hypoxic tolerance between normal two triploids (4_??_×2_??_and 2_??_×4_??_). However, significant differences were observed in the mortalities in bacterial gill disease (BGD) -infected triploids. The tolerance to BGD in triploid (4_??_×2_??_) was higher than those in triploid (RSP) and triploid (2_??_×4_??_). Since it is known that fish with BGD die from asphyxiation and its mortality is affected by the environmental oxygen level, hemoglobin concentration of fish would be closely associated with the tolerance against BGD.
Two studies were designed to quantify the effect of water temperature and flow rate on the transmission potential of the important salmonid gill pathogen, Loma salmonae. Using survival analysis, increased water temperature and low flow rates were determined as risk factors for the transmission of microsporidial gill disease caused by L. salmonae in rainbow trout Oncorhynchus mykiss. Fish were experimentally infected with L. salmonae via a cohabitation exposure model and monitored for the development of branchial xenomas. On any given day, fish held at 11°C and 15°C had a hazard ratio equal to 0.80 and 0.68, respectively, for the development of branchial xenomas compared with fish held at 19°C. From the flow rate trial, fish housed in a low flow tank (0.83 L/min) had an increased chance of developing branchial xenomas when compared to fish in tanks at normal (1.67 L/min) and high (2.5 L/min) flow rates with hazard ratios reported as 0.69.
Susceptibilities of six species of marine fish to a flounder isolate KRRV-9601 (a European genotype) of VHSV were examined by intraperitoneal injection. Mortalities were induced in Japanese flounder Paralichthys olivaceus (100%), black sea bream Acanthopagrus schlegeli (90%), red spotted grouper Epinephelus akaara (70%) and rockfish Sebastes schlegeli (10%), but not in marbled sole Pleuronectes yokohamae and red sea bream Pagrus major. VHSV was recovered from dead and moribund fish and the surviving red spotted grouper at 60th day after the virus injection. The neutralizing antibody against VHSV was detected in the sera of surviving rockfish, but not in other surviving fishes. Histopathological features in the affected fish for each species were generally similar to each other, although the severity of hemorrhage varied among fish species.
Rapid in situ hybridization (ISH) technique was developed for the detection of fish microsporidian parasites. All developmental stages of Glugea plecoglossi including the initial stage immediately after entry into the fish tissue could be detected by rapid ISH using DIG-labelled oligonucleotide probes. However, ISH signals were only faintly visible from mature spores of Microsporidium seriolae, while the pre-sporogonic stages were clearly positive.Thus, it is recommended to use both rapid ISH and Uvitex 2B stain for the detection of all stages of M. seriolae. The rapid ISH allows the whole process to be completed within 2h, and thus this technique is both convenient and practical as a diagnostic and research tool for studying the life cycle of microsporidians.