Fish Pathology Vol. 49(2014) No. 4

Vaccination with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of Edwardsiella tarda has been demonstrated to have cross-protection against some other bacterial pathogens. We prepared a recombinant protein and pcDNA4 expression vector of GAPDH of E. tarda and injected those to 0-age yellowtail Seriola quinqueradiata to evaluate their protective efficacies against Nocardia seriolae. The 60-d survival rates ranged from 0% to 6.7% among vaccinated and control groups, suggesting that the protein and expression vector of E. tarda GAPDH had no protective effect against nocardiosis. However, the copy numbers of the 16S ribosomal RNA gene of N. seriolae were significantly lower in the gill, kidney, and spleen of vaccinated fish 9 and 18 days post-infection compared with those in the control group that was also challenged with N. seriolae. Additionally, the CC chemokine, major histocompatibility complex class II α antigen, interleukin 1β, and immunoglobulin M genes were significantly upregulated in the vaccinated groups. Four predicted B-cell epitopes of GAPDH in E. tarda, which may play an important role in cross-protection, differed in amino acid sequences from those of GAPDH in N. seriolae. This may explain the lack of a protective effect of E. tarda GAPDH against N. seriolae.

Acute hepatopancreatic necrosis disease (AHPND) is caused by a unique strain of Vibrio parahaemolyticus that has a plasmid harboring virulent genes. A rapid and accurate diagnosis method is necessary for surveillance of this infectious disease in aquaculture. In this study, three primer sets (TUMSAT-Vp1, Vp2 and Vp3) were designed based on the plasmid DNA sequence. We examined 98 strains of bacteria isolated from shrimp farms in different areas in Thailand. These included 48 strains of V. parahaemolyticus (AHPND strain), 38 strains of non-AHPND V. parahaemolyticus and 12 strains of non-V. parahaemolyticus. All the AHPND strains were detected by TUMSAT-Vp1, Vp2 and Vp3. However, one non-AHPND strain tested positive for TUMSAT-Vp1 and Vp2. The accuracy of AHPND detection was validated with two other primer publicly available sets (AP1 and AP2). AP1 and AP2 primers gave a few false positives in non-AHPND strains. Only TUMSAT-Vp3 primer detected the AHPND strains examined in this study with 100% accuracy.

This study presents the development of DNA vaccines against nocardiosis, using the Antigen 85-like (Ag85L) gene in Nocardia seriolae. An expression plasmid encoding Ag85L (pAg85L^wt) and codon-optimized Ag85L (pAg85L^opt) was intramuscularly injected into amberjack Seriola dumerili. Survival rates of the pAg85L^wt, pAg85L^opt vaccinated group and PBS-injected group as a negative control were 88%, 98% and 51%, respectively, at 40 days after the N. seriolae challenge. In addition, the N. seriolae bacterial count in the spleen was significantly lower in the pAg85L^wt and pAg85L^opt vaccinated fish than in the PBS-injected fish (P < 0.05). These results suggest that the DNA vaccines pAg85L^wt and pAg85L^opt conferred protective efficacy against N. seriolae infection in amberjack.

The advancement in nucleic acid amplification has improved the diagnostic methods for many diseases. In the current study, an improved technique for the detection of infectious spleen and kidney necrosis virus (ISKNV) based on loop-mediated isothermal amplification (LAMP) is described. The addition of acridine orange at the end of the reaction causes distinct color change which can be recognized by the naked eye. In addition, a new set of primers was designed based on the ISKNV major capsid protein (MCP) gene sequences. The primers are highly specific for ISKNV and there was no cross amplification with red sea bream iridovirus (RSIV), white spot syndrome virus (WSSV), Aeromonas hydrophila or Vibrio parahaemolyticus. The detection limit of LAMP assay was 1.4 × 10⁴ copies of the virus DNA and the optimum temperature and time for assay was 65°C and 60 min respectively, and this protocol could successfully detect the virus from asymptomatically infected fish. This improved LAMP assay is a simple and inexpensive diagnostic tool for the detection of ISKNV without the need of specialized equipment.

A paramyxean parasite infection was observed in the epithelial cells of the stomach, intestine, and digestive diverticula of Manila clam Ruditapes philippinarum collected from Odawa Bay, Japan. The mean detection frequency between April 2010 and September 2011 as revealed by histology was 8.9% with a maximum monthly frequency of 28.6%, but remarkable mass mortality was not recognized during the study period. Sporulation of the parasites involves repeated production of intracellular cells, and a primary cell contains 8 secondary cells, in which 4 multicellular spore cells are produced. Several large eosinophilic granules formed in the cytoplasm of secondary cells during the final maturation process. Transmission electron microscopy (TEM) revealed that spore cells consisted of the innermost, intermediate and outermost cells, indicating that this parasite is a member of the genus Marteilia. TEM observations also revealed that the cell membrane of mature spores was lined with an electron-dense monolayer, which has not been reported from other species in the genus Marteilia. Determined SSU rRNA gene sequence of this parasite was apparently different from previously described Marteilia species, and this species was identified as Marteilia granula n. sp., a new species in the genus Marteilia, Order Paramyxida, Phylum Cercozoa. Our results identify for the first time the parasite species of the genus Marteilia infecting the Manila clam in Japan.

We investigated the applicable organ to detect DNA of cyprinid herpesvirus 3 (CyHV-3), the causative agent of koi herpesvirus disease (KHVD). CyHV-3 DNA was detected at the highest positive rate in the caudal fin among several organs including gills, kidney and spleen of the fish sequentially sampled after experimental exposure to the virus. Moreover, CyHV-3 DNA was detected in the caudal and pectoral fins of all dead fish collected in a natural KHVD case. Since fins are an easily accessible organ, they can be an applicable organ for PCR-based diagnosis of KHVD in clinical fish.

We investigated the existence of Diphyllobothrium nihonkaiense plerocercoids and trematode metacercariae of the genus Metagonimus in salmonid fishes (N = 2,187) cultured in freshwater areas of Japan. The infection of D. nihonkaiense plerocercoid larvae was not detected in cultured freshwater salmonids, whilst five out of 60 wild anadromous masu salmon Oncorhynchus masou masou captured during the spawning migration in the Miomote River was infected with D. nihonkaiense. No Metagonimus metacercariae were detected in any salmonids examined. The present study corroborates the previous report indicating that salmonids cultured in freshwater areas of Japan have no chance of infection with D. nihonkaiense.

In November 2011, the renal sphaerosporosis was found in cage-cultured orange-spotted grouper Epinephelus coioides in a fish farm in Guangdong, China. The infected fish exhibited emaciation, anaemia, anorexia, renomegaly, slight splenomegaly, and occasionally skin ulcer. The cumulative mortality reached at 50% to 80% within 2 weeks, when water temperature ranged from 21°C to 27°C. The renal tubules were almost completely occluded by sporogonic pseudoplasmodia of a myxosporean, and mature spores were found in the lumen. Combined with morphological, histopathological, and molecular analyses, Sphaerospora epinepheli was suggested as the etiological agent. This is the first report of mortality case of cultured E. coioides associated with S. epinepheli.

The stalked sea squirt, Styela clava, was examined for the presence of the kinetoplastid, Azumiobodo hoyamushi, the causative agent of soft tunic syndrome in the edible ascidian, Halocynthia roretzi. Apparently healthy S. clava individuals were collected from a H. roretzi culture site, which was epizootic area of A. hoyamushi. After rearing for 50 days, the tunics of seven out of 48 specimens became softened and were found infected with A. hoyamushi. Healthy H. roretzi were experimentally infected with A. hoyamushi by rearing in water containing tunic from the diseased S. clava. Our results indicate that S. clava is a potential carrier of A. hoyamushi.