Comparative transcriptome analyses of the third and fourth stage larvae of Anisakis simplex (Nematoda: Anisakidae).

We analyzed transcriptome profiles of Anisakis simplex (Nematoda: Anisakidae) 3rd (ASL3) and 4th larvae (ASL4) obtained by RNA-seq, to understand the molecular pathways linked to parasite survival and discover stage-enriched gene expressions. ASL3 were collected from chum salmon and ASL4 were obtained by in vitro culture. Whole transcriptome sequencing was conducted with Illumina sequencer, and de novo assembly was conducted. 47,179 and 41,934 genes were expressed in ASL3 and ASL4 transcriptomes. Of them, 17,633 were known and 29,546 were unmapped sequence for ASL3. 17,126 were known and 24,808 were unmapped sequence for ASL4. Polyubiquitins-related genes and collagen-related genes were the most abundantly expressed in ASL3 and ASL4. Mitochondrial enzyme-related genes were highly expressed both in ASL3 and ASL4. Among the transcripts, 675 were up-regulated in ASL3, while 1015 were up-regulated in ASL4. Several protease-related and protein biosynthesis-related genes were highly expressed in ASL3, all of which are thought to be crucial for invading host tissues. Collagen synthesis-related genes were highly expressed in ASL4, reflecting active biosynthesis of collagens during molting process. This information will extend our understanding of biology of the fish-borne zoonotic parasite A. simplex.

Development of PCR method for detecting Kudoa iwatai (Myxozoa: Multivalvulida) from rock bream Oplegnathus fasciatus.

We developed a PCR assay targeting the 28S rDNA of Kudoa iwatai (Multivalvulida: Myxozoa) and investigated the prevalence of infection in rock bream Oplegnathus fasciatus, which is commercially an important aquaculture species in Korea, with this assay. Detection limit of the PCR assay was 2.5 fg/µl with plasmid DNA and 8.6 × 103 spores/ml with purified spores, respectively. This PCR assay did not amplify DNA of other Kudoa species (Kudoa septempunctata, Kudoa lateolabracis, Kudoa thyrsites) tested. Sliced muscles of whole body from 318 rock bream (wild and cultured) were examined by this PCR assay and also with the naked eyes. All of the wild fish did not produce amplicons nor did harbor visible Kudoa cysts (0/70). Three of the cultured fish were PCR-positive and also harbored visible Kudoa cysts (3/248, 1.2%). The sequences of amplicons (574 bp) were 100% identical with those of the K. iwatai already registered in Genbank. When the visceral organs of these three fish were examined, visible cysts were not found, but one stomach sample was found to be PCR-positive. There was no difference in the prevalence of infection estimated by PCR assay and the presence of visible Kudoa cysts in our samples. This is thought to be because the development of K. iwatai is already completed and only mature Kudoa cysts existed in our samples.

Pathogenic potential of two sibling species, Anisakis simplex (s.s.) and Anisakis pegreffii (Nematoda: Anisakidae): in vitro and in vivo studies.

The pathogenic potentials of two sibling nematodes Anisakis simplex sensu stricto (s.s.) and A. pegreffii were compared by in vitro and in vivo studies. Live third-stage larvae of each species were subjected to agar blocks made using PBS or RPMI-1640, overlaid with different supernatants (artificial gastric juice, PBS, and RPMI-1640), and their penetration ability was compared. Their tolerance of artificial gastric juice was also tested. Further, they were introduced into rats by gastric intubation, and the in vivo locations of them were investigated. A. pegreffii showed higher penetration ability than A. simplex (s.s.) in most of the experimental conditions, except for the RPMI-1640 agar block overlaid with artificial gastric juice. In an acid tolerance test, the mean survival times were 6.1 days for A. simplex (s.s.) and 4.2 days for A. pegreffii. In an animal experiment, A. simplex (s.s.) stayed for a shorter time in the stomachs of rats than A. pegreffii. Some A. pegreffii and A. simplex (s.s.) were embedded in the gastric mucosa or freely existed in the abdominal cavity. All of these results suggest that A. pegreffii has the pathogenic potential to cause anisakidosis in humans when ingested, as does A. simplex (s.s.).

Occurrence of anisakid nematode larvae in chub mackerel (Scomber japonicus) caught off Korea.

Chub mackerel (Scomber japonicus) is a pelagic fish species widely distributing in the Indo-Pacific and a commercially important fish species in Korea. It is known to harbor anisakid nematodes larvae, and ingesting the raw or undercooked fish can accidentally cause human infection. In this study, we isolated the nematode larvae in 417 chub mackerel caught from 7 sampling locations around the Korean Peninsula in 2011 and 2012, and identified them by PCR-RFLP of the ITS (internal transcribed spacer) of ribosomal DNA and the direct sequencing of the mitochondrial DNA cox2 gene. The prevalence of infection was 55.4% (231/417) and the mean intensity was 7.0 (1628/231). Most of the nematodes (1523/1628; 93.6%) were found in the body cavity, while 5.5% (89/1628) were found in the gastrointestinal tract. Four different species were identified by PCR-RFLP and direct sequencing. Most of the nematodes (1535/1628; 94.3%) were identified as Anisakis pegreffii, and 2.8% (46/1628) were identified as Hysterothylacium sp. A hybrid genotype (Anisakis simplex sensu stricto×A. pegreffii) and A. simplex sensu stricto were 2.5% (41/1628) and 0.4% (6/1628) of the identified nematodes, respectively. The anisakid nematode assemblage of chub mackerel in Korea was similar to that of chub mackerel from the Tsushima Current stock in Japan, in that A. pegreffii was the dominant species. Since most of the anisakid nematodes were found in the body cavity and most of them were identified as A. pegreffii or Hysterothylacium sp. by PCR-RFLP and direct sequencing, chub mackerel may not greatly contribute to human anisakidosis in Korea. Alternately, A. pegreffii may be responsible for human anisakidosis in Korea, in addition to A. simplex sensu stricto. Further studies, such as the molecular diagnosis of human anisakidosis, are necessary for assessing the epidemiological role of chub mackerel in Korea.

Development of loop-mediated isothermal amplification method for detection of Kudoa septempunctata (Myxozoa: Multivalvulida) in olive flounder (Paralichthys olivaceus).

A loop-mediated isothermal amplification (LAMP) assay was developed and validated for early, rapid, and sensitive detection of Kudoa septempunctata, a myxosporean parasite found in olive flounder (Paralichthys olivaceus). Recently, several outbreaks associated with ingestion of raw olive flounder muscles harboring mature K. septempunctata spores have been reported, and it is becoming obvious that fresh K. septempunctata spores can cause problems in humans when ingested. Thus, it is necessary to develop reliable detection method of K. septempunctata, to prevent outbreaks and ensure food safety. The LAMP assay has advantages over other molecular detection methods for detecting K. septempunctata in olive flounder muscle, in terms of simplicity, rapidity, and sensitivity. The reaction condition was optimized as 63 °C, 45 min, with three sets of specific primers. The results can be simply confirmed with the naked eye after adding SYBR Green I or by conventional electrophoresis followed by ethidium bromide staining. This LAMP assay did not show any cross-reaction with other kudoid myxosporeans (Kudoa lateolabracis, Kudoa thyrsites) can be found in olive flounder muscles and was validated by testing Kudoa septempunctata spore-spiked samples and field samples. The results showed that the LAMP assay is ten times more sensitive than the conventional polymerase chain reaction in this study and can be applied for early detection for monitoring and epidemiological studies of K. septempunctata in olive flounder aquaculture farms.

Reverse transcriptase loop-mediated isothermal amplification assay for infectious hematopoietic necrosis virus in Oncorhynchus keta.

A reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay was developed for detecting infectious hematopoietic necrosis virus (IHNV) from chum salmon Oncorhynchus keta in South Korea with high specificity, sensitivity and rapidity. A set of 6 IHNV-specific primers was designed, based on the G-protein sequence of IHNV (PRT strain), recognizing 8 distinct sequences of the target RNA. The assay was optimized to detect IHNV at 63 degrees C for 30 min. The limit of detection was 0.01 fg of RNA extracted from IHNV-infected CHSE-214 cells, compared with 1.0 fg for nested RT-PCR. The applicability of this RT-LAMP assay was further tested by comparison with nested RT-PCR using field samples. Of 473 samples tested, 191 samples (40.38%) were IHNV-positive by RT-LAMP, whereas 162 samples (34.25%) were IHNV-positive by nested RT-PCR. These results indicate that, because of its high sensitivity and rapidity, the RT-LAMP assay is useful for early diagnosis of IHN.

Occurrence and identification of Anisakis spp. (Nematoda: Anisakidae) isolated from chum salmon (Oncorhynchus keta) in Korea.

The prevalence of infection and the identification of anisakid larvae in chum salmon (Oncorhynchus keta) from the Namdae River, the east coast of Korea, were investigated. In total, 8,358 larvae were collected from 120 fish samples (male = 58 fish, female = 62 fish) in 2008. Fish samples were collected during October and November 2008. All the chum salmon samples (120/120, 100%) caught were infected with anisakid larvae with a high intensity (69.65 ± 48.58 larvae/host). They were mostly found in muscles (98.00%). Based on the morphological and the molecular analysis of PCR-RFLP and sequencing of mitochondrial DNA cox2 gene markers, these nematodes were identified as Anisakis simplex (sensu stricto) third-stage larvae. This is the first report on the molecular identification of anisakid worms from salmonid fishes in Korea. The high occurrence of anisakid worms in chum salmon may pose considerable food safety problems if they were consumed as raw or undercooked, although their commercial value is relatively lower than other salmonid species.

Detection of megalocytivirus from imported tropical ornamental fish, paradise fish Macropodus opercularis.

Megalocytivirus was detected from paradise fish Macropodus opercularis imported from Indonesia. Four of 11 fish (36%) in 2006 and 40 of 117 fish (34%) in 2008 were found to be PCR-positive for megalocytivirus. Phylogenetic analysis based on partial major capsid protein (MCP) gene nucleotide sequences revealed that the sequences detected in paradise fish were classified as Genotype II, which includes freshwater fish isolates from Southeast Asian countries, closely related to infectious spleen and kidney necrosis virus (ISKNV), Murray cod iridovirus (MCIV), and dwarf gourami iridovirus (DGIV-2004). Paradise fish was added as a new host for megalocytivirus based on this study.