Withering syndrome (WS) is a chronic wasting disease in abalone caused by ‘Candidatus Xenohaliotis californiensis’, a Rickettsia-like organism (RLO). Japanese black abalone Haliotis discus discus (14-34 mm in shell length) experienced monthly mortality rates of 3 to 10% from September 2010 to January 2011 with a cumulative mortality of 33% in a hatchery. Nine moribund animals were examined: five animals by histopathology and four by PCR. In histopathological examination, three animals exhibited basophilic, intracellular bacterial inclusions in the epithelium of the digestive tract. Severe infection was observed in the epithelium of the posterior portion of the esophagus of one animal. Morphological characteristics, and target tissues containing the inclusions were consistent with WS-RLO infection. In a PCR for 16S rRNA gene of WS-RLO, a specific fragment was amplified from all four animals, and nucleotide sequences of the amplicons were identical to that of the WS-RLO reported previously. The RLOs observed were conclusively identified as the agent of WS. Despite infection with the WS-RLO, our observations showed little degeneration of the digestive gland and shrinkage of the foot muscle both of which are critical pathological changes in WS. This is the first report of the WS-RLO found in Japanese black abalone in Japan.
PCR assays were developed for the rapid and sensitive detection of Azumiobodo hoyamushi, the protistan pathogen that causes soft tunic syndrome in the cultured ascidian Halocynthia roretzi. Two sets of A. hoyamushi-specific primers based on the 18S rRNA and β-tubulin gene sequences, respectively, of the flagellate were designed and validated for the detection of A. hoyamushi. In both PCR assays, the flagellate genes were detected in diseased ascidians, but not in apparently healthy ones. When DNA were extracted from flagellate suspensions obtained from infected tunics, the detection limits of the PCR assays using primers for the 18S rRNA and β-tubulin were 3.4 × 10 cells/mL and 3.4 × 102 cells/mL, respectively. Even at the early stage of infection when the slight clinical signs were observed only in the siphons, A. hoyamushi was detected from the affected tissue using 18S rRNA PCR, but not β-tubulin PCR. Thus, the 18S rRNA PCR is a rapid, sensitive and specific assay for diagnosing soft tunic syndrome.
Hemorrhagic disease occurred in cultured fingerlings of snakehead Channa striata in Viet Nam. Bacteria isolated from affected fish were identified as Aeromonus hydrophila. One of the isolates from lesions of a moribund cultured snakehead fingerling was subjected to a pathogenicity test to snakehead fingerling using intraperitoneal injection. Inoculation with the bacterium caused clinical signs in challenged fingerlings similar to those observed in naturally affected fish. Resulting mortality indicated an LD50 of 1.2 × 105 CFU/fish. Molecular characterization demonstrated that the same bacterium was re-isolated from the experimentally infected fish. This is the first report of A. hydrophila infection in snakehead in Viet Nam.
We attempted to assess the risk that goldfish Carassius auratus potentially transmits KHV to naïve carp Cyprinus carpio. Goldfish including three varieties and koi carp were exposed to three KHV isolates from Japan, USA and Israel, and cohabited with recipient naïve ‘wild type’ common carp, a highly susceptible strain, from 2 to 24 days post viral exposure (dpe). All of the recipient carp cohabited with KHV-exposed koi carp died due to KHV infection, whereas no mortality was observed in the recipient carp cohabited with the exposed goldfish. In addition, no KHV was detected in organs of the goldfish at 24 dpe by PCR. These results indicate that goldfish is not a susceptible host.
We designed a universal primer set for quantitative polymerase chain reaction (qPCR) based on the nucleotide sequences of several fish β-actin genes for counting cell number in small amounts of fish tissue. We also evaluated primer reactivity using several established fish cell lines and blood cells from diverse fish species. A strong correlation was observed between cell numbers and Ct values among olive flounder, sevenband grouper, rock bream, rock fish, rainbow trout and fish cell lines (FHM, EPC, SSN-1, BF-2 and GF), suggesting that cell number can be counted by qPCR assay using the universal primers for a wide range of fish species.