Two human cases of gnathostomiasis and discovery of a second intermediate host of Gnathostoma nipponicum in Japan

Two human cases of gnathostomiasis from ingestion of raw native Japanese loaches, Misgurnus anguillicaudatus, are reported. Seven early third-stage larval Gnathostoma nipponicum were recovered from 3,098 loaches in the same district in which 2 human patients had obtained and eaten raw loaches. Encapsulated G. nipponicum larvae were also recovered from loaches infected under laboratory conditions. All 6 weasels captured in the same district in which the naturally infected loaches were found and where the humans had become infected were infected with adult worms of the same species. This is the first report of M. anguillicaudatus serving as a second intermediate host of G. nipponicum.     

A human case of gnathostomiasis nipponica confirmed indirectly by finding infective larvae in leftover largemouth bass meat

A human case of creeping eruption due to Gnathostoma nipponicum was confirmed indirectly by finding infective advanced third-stage larvae in leftover largemouth bass meat. This is the first report indicating that the largemouth bass (Micropterus salmoides) serves as a source of G. nipponicum infection in humans.     

Ultrastructure and functions of the cells of proximal and distal segments of the nephron of anadromous and freshwater fishes]

In the carp kidney, the proximal tubule cells have no infoldings of the basilar plasmatic membrane. In the Salmonidae nephrons the numerous parallel membranes of proximal tubule cell are formed by foldings of the basilar plasmatic membrane with numerous mitochondria in between. In O. nerka and S. malma experiments with MgCl2 injection showed that their kidneys secrete magnesium in contrast to the carp kidney. The numerous invaginations of proximal tubule cells membrane are suggested to be a morphological equivalent of the Na/Mg exchange mechanism.     

Discovery of larval Gnathostoma nipponicum in frogs and snakes from Jeju-do (Province), Republic of Korea

A survey was performed to find out the intermediate hosts of Gnathostoma nipponicum in Jeju-do (Province), the Republic of Korea. In August 2009 and 2010, a total of 82 tadpoles, 23 black-spotted pond frogs (Rana nigromaculata), 7 tiger keelback snakes (Rhabdophis tigrinus tigrinus), 6 red-tongue viper snakes (Agkistrodon ussuriensis), and 2 cat snakes (Elaphe dione) were collected in Jeju-do and examined by the pepsin-HCl digestion method. Total 5 gnathostome larvae were detected in 3 (50%) of 6 A. ussuriensis, 70 larvae in 3 of 7 (42.9%) R. tigrinus tigrinus, and 2 larvae in 2 of 82 (8.7%) frogs. No gnathostome larvae were detected in tadpoles and cat snakes. The larvae detected were a single species, and 2.17 × 0.22 mm in average size. They had characteristic head bulbs, muscular esophagus, and 4 cervical sacs. Three rows of hooklets were arranged in the head bulbs, and the number of hooklets in each row was 29, 33, and 36 posteriorly. All these characters were consistent with the advanced third-stage larvae of G. nipponicum. It has been first confirmed in Jeju-do that R. nigromaculata, A. ussuriensis, and R. tigrinus tigrinus play a role for intermediate and/or paratenic hosts for G. nipponicum.     

Differences in host selection of actinospores of two myxosporeans, Myxobolus arcticus and Thelohanellus hovorkai

We investigated the host selection mechanism of actinospore stages of 2 myxosporeans, Myxobolus arcticus and Thelohanellus hovorkai, infecting masu salmon (Oncorhynchus masou) and common carp (Cyprinus carpio), respectively. Discharge of the polar filaments and sporoplasm release by M. arcticus actinospores occurred within the first 5 min of exposure to skin mucus of masu salmon. The actinospores also reacted to the mucus of nonsusceptible fish, i.e., sockeye salmon (Oncorhynchus nerka) and goldfish (Carassius auratus), although the reactivity was comparatively lower. After exposure of masu, and sockeye and chum salmon (Oncorhynchus keta) to M. arcticus actinospores, the penetration of sporoplasms was observed in the fins and gills of masu and sockeye salmon to a similar extent and to a lesser extent in chum salmon. Thelohanellus hovorkai actinospores exhibited a slow response of sporoplasm release to common carp mucus as well as penetration into the gills of common carp. Neither chemoresponse to mucus of nonsusceptible fish (goldfish and sockeye salmon) nor sporoplasm invasion in goldfish was observed for T. hovorkai actinospores. These results indicate notable differences in the host selection at the time of entry between M. arcticus and T. hovorkai; the former responds quickly to fish mucus with low host specificity, whereas the latter was highly host specific in a dilatory reaction.     

Life cycle of Gnathostoma nipponicum Yamaguti, 1941.

The life cycle of Gnathostoma nipponicum was examined by field survey and by experimental infection of animals with the larvae. Naturally infected larval G. nipponicum were found in loaches, catfish, and snakes. Experimentally, loaches, killifishes, frogs, salamanders, mice, and rats were successfully infected with the early third-stage larvae of G. nipponicum obtained from copepods (the first intermediate host), whereas snakes, quails, and weasels were not. Frogs, snakes, quails, and rats were experimentally infected with the advanced third-stage larvae (AdL3) from loaches. These results reveal that some species of fishes, amphibians and mammals can act as the second intermediate host and that some species of reptiles, birds and mammals can act as a paratenic host. The life cycle was completed in weasels, the definitive host, which were infected with AdL3 from loaches and started to evacuate eggs of G. nipponicun in faeces on days 65-90 postinfection.     

Molecular cloning and expression analysis of a novel caspase recruitment domain protein (CARD) in common carp Cyprinus carpio L

A novel caspase recruitment domain protein (CARD) was isolated from common carp Cyprinus carpio L. by expressed sequence tag analysis. This gene consist of a 2016 bp open reading frame and untranslated regions, which is putatively translated to a protein of 535 amino acid residues. The gene harbors domains (CARD and Coiled-coil domain), which are conserved in proteins of CARD family. The CARD domain have carp was similar to human CARD9 with 72.4% identity. Expression analysis revealed that CARD gene of carp (carp-CARD) expressed in normal tissues of head kidney, spleen, liver, heart and brain. Here we demonstrated that the expression of carp-CARD increased by cortisol treatment in all the tissues and had a high and long lasting expression in cortisol treated spleen.     

Imported intraocular gnathostomiasis with subretinal tracks confirmed by western blot assay

We report a case of intraocular gnathostomiasis diagnosed by western blot assay in a patient with subretinal tracks. A 15-year-old male patient complained of blurred vision in the right eye, lasting for 2 weeks. Eight months earlier, he had traveled to Vietnam for 1 week and ate raw wild boar meat and lobster. His best-corrected visual acuity was 20/20 in both eyes and anterior chamber examination revealed no abnormalities. Fundus examination showed subretinal tracks in the right eye. Fluorescein angiography and indocyanine green angiography showed linear hyperfluorescence of the subretinal lesion observed on fundus in the right eye. Ultrasound examination revealed no abnormalities. Blood tests indicated mild eosinophilia (7.5%), and there was no abnormality found by systemic examinations. Two years later, the patient visited our department again for ophthalmologic evaluation. Visual acuity remained 20/20 in both eyes and the subretinal tracks in the right eye had not changed since the previous examination. Serologic examination was performed to provide a more accurate diagnosis, and the patient's serum reacted strongly to the Gnathostoma nipponicum antigen by western blot assay, which led to a diagnosis of intraocular gnathostomiasis. This is the first reported case of intraocular gnathostomiasis with subretinal tracks confirmed serologically using western blot in Korea.     

Prevalence of larval Anisakis simplex in pen-reared and wild-caught salmon (Salmonidae) from Puget Sound, Washington

The abundance of parasites of public health significance in pen-reared salmon and wild-caught salmon was compared. Two hundred eighty-seven salmon from Puget Sound, Washington, were examined for third-stage larvae of Anisakis simplex. Of these fish, 237 Atlantic salmon (Salmo salar), coho salmon (Oncorhynchus kisutch) and chinook salmon (O. tshawytscha) were reared in commercial salmon pens and 50 sockeye salmon (O. nerka) were caught during their spawning migration. All wild-caught salmon were found to be infected with larval A. simplex; conversely, all pen-reared fishes lacked such infections. Edible musculature of wild salmon were infected with 581 (87%) nematode larvae. Of other salmon parasites known to infect humans, one Diphyllobothrium sp. plerocercoid was collected from each of three of the 50 wild-caught salmon. The study showed that farmed salmon may increase the margin of safety for consumers of raw seafood.     

Molecular cloning and characterization of alpha-class glutathione S-transferase gene from the liver of silver carp, bighead carp, and other major Chinese freshwater fishes

Two full-length cDNAs encoding glutathione S-transferase (GST) were cloned and sequenced from the hepatopancreas of planktivorous silver carp (Hypophthalmichthys molitrix) and bighead carp (Aristichthys nobilis). The silver carp and bighead carp GST cDNA were 920 and 978 bp in length, respectively, and both contained an open reading frame that encoding 223 amino acids. Partial GST cDNA sequences were also obtained from the liver of grass carp (Ctenopharyngodon idellus), crucian carp (Carassius auratu), mud carp (Cirrhinus molitorella), and tilapia (Oreochromis nilotica). All these GSTs could be classified as alpha-class GSTs on the basis of their amino acid sequence identity with other species. The three-dimensional structure of the silver carp GST was predicted using a computer program, and was found to fit the classical two-domain GST structure. Using the genome walker method, a 875-bp 5'-flanking region of the silver carp GST gene was obtained, and several lipopolysaccharide (LPS) response elements were identified in the promoter region of the phytoplanktivorous fish GST gene, indicating that the GST gene expression of this fish might be regulated by LPS, released from the toxic blue-green algae producing microcystins. To compare the constitutive expression level of the liver GST gene among the six freshwater fishes with completely different tolerance to microcystins, beta-actin was used as control and the ratio GST/beta-actin mRNA (%) was determined as 130.7 +/- 6.6 (grass carp), 103.1 +/- 8.9 (bighead carp), 92.6 +/- 15.0 (crucian carp), 72.3 +/- 7.8 (mud carp), 58.8 +/- 11.5 (silver carp), and 33.6 +/- 13.7 (tilapia). The constitutive expression level of the liver GST gene clearly shows that all the six freshwater fishes had a negative relationship with their tolerance to microcystins: high-resistant fishes (phytoplanktivorous silver carp and tilapia) had the lowest tolerance to microcystins and the high-sensitive fish (herbivorous grass carp) had the highest tolerance to microcystins. Taken together with the reciprocal relationship of constitutive and inducible liver GST expression level in some of the tested fish species to microcystin exposure, a molecular mechanism for different microcystin detoxification abilities of the warm freshwater fishes was discussed.     

Sex chromosomes in the sockeye salmon: a Y-autosome fusion

Chromosomes of 21 sockeye salmon [Oncorhynchus nerka (Walbaum)] from three locations in Washington state were examined. All males had 57 chromosomes, while all females had 58 chromosomes. Both sexes had 104 chromosome arms. It appears that in males of this species the Y chromosome and an autosome have fused to form a metacentric chromosome.     

A mass occurrence of human infection with Diplogonoporus grandis (Cestoda: Diphyllobothriidae) in Shizuoka Prefecture, central Japan

Forty-six cases of human infection with Diplogonoporus grandis were found in Shizuoka Prefecture on the Pacific coast of Central Japan in 1996. The cases were predominantly elderly male patients over 50 years of age. Although all cases were reported from May to September of the year, most of them were diagnosed in June and July. We suspected that the transmission was due to the consumption of raw juvenile Japanese anchovy (Engraulis japonicus), which are seasonally caught in the spring months off the Pacific coast of the Prefecture. In almost a hundred years after its discovery in 1894, there had been more than 180 cases of human diplogonoporiasis recorded in Japan. The high incidence within a relatively short time frame of our investigation is regarded unusual even in this country.     

草鱼连续两代的雌核发育群体及湘江流域群体基因组DNA的微卫星比较分析

对湘江流域草鱼群体,一代及连续两代极体型人工诱导雌核发育草鱼群体基因组DNA的微卫星引物统计分析结果表明:湘江流域草鱼群体存在一定程度的遗传多态性;大多数被检测的微卫星位点上存在两个以上的等位基因,但遗传多样性程度较低。一代人工诱导雌核发育草鱼群体中个体的基因位点已基本纯合,但就整个群体而言,个体之间的基因型还不完全一致,表现出一定的多态性。连续两代人工诱导雌核发育草鱼群体中不仅所检测个体的基因位点已完全纯合,并且各个体的基因型也完全相同。这些观察结果说明所检测的两代人工诱导雌核发育草鱼群体是纯合的,经连续两代人工诱导雌核发育可能建立起草鱼纯系。该实验结果还发现不仅草鱼的微卫星位点上存在等位基因的多态性,而且微卫星位点本身也存在多态性;在人工诱导草鱼雌核发育的过程中不仅存在微卫星等位基因快速丢失的现象,而且也存在微卫星位点丢失的现象。因此,加强对自然水体中草鱼种质资源多样性的保护和利用各种现代生物学技术纯化、筛选和组合优良性状基因,是草鱼遗传育种中同样重要和不可或缺的两个方面。     

Detection of infection and susceptibility of different Pacific salmon stocks (Oncorhynchus spp.) to the haemoflagellate Cryptobia salmositica

The haematocrit centrifugation technique, modified by keeping the haematocrit tubes cold (between 1 and 10 C), was sensitive for detecting light infections of Cryptobia salmositica (as few as 75 flagellates per ml of blood). In wet mount preparations, infections lighter than 7.5 X 10(3) flagellates per ml of blood could not be detected consistently. Different Pacific salmon stocks from British Columbia demonstrated differences in susceptibility to C. salmositica in experimental studies using laboratory reared juvenile fish. Oncorhynchus keta and Oncorhynchus tshawytscha from the Big Qualicum River stocks (Vancouver Island), and Oncorhynchus nerka from the Fulton River stock (Skeena River system), were all equally susceptible and suffered high mortalities at low exposures (100 flagellates in 0.1 ml physiological saline inoculated intraperitoneally per fish). Oncorhynchus nerka from the Weaver Creek stock (Fraser River system) was the most resistant with no mortalities even at exposures of 10(6) flagellates (in 0.1 ml physiological saline) per fish. Oncorhynchus kisutch seemed to be slightly less resistant than the Weaver Creek O. nerka, but fewer than 16% of the inoculated fish died. Oncorhynchus kisutch from the Big Qualicum River seemed to be slightly more resistant than O. kisutch from the Capilano River stock (a coastal river near Vancouver), with fewer mortalities and lighter infections when the experiments were terminated. Differences in susceptibility are believed to be associated with innate, genetically transmitted resistance.     

Surface ultrastructure of the advanced third-stage larvae of Gnathostoma nipponicum

The surface ultrastructure of advanced third-stage larvae (AL3) of Gnathostoma nipponicum was studied using scanning electron microscopy. The larvae were recovered from the grass snake Rhabdophis tigrina in the Republic of Korea. Parasites had a globular head bulb with a pair of lips at the anterior end and 2 labial papillae and an amphid on each lip. The head bulb was characteristically armed with 3 transverse rows of hooklets, averaging 36, 38, and 43 in number, increasing posteriorly. A total of 213-232 minute unidentate cuticular spines were present along the entire length of the larvae, forming the transverse striations. Two pairs of cervical papillae were located between the 8th and 12th transverse striations, and a pair of body papillae was seen laterally on the posterior third of the body. A pair of caudal phasmids was recognized near the posterior extremity. The surface ultrastructure of AL3 of G. nipponicum is unique compared with that of other species.     

Structural and immunochemical studies of carp (Cyprinus carpio L.) immunoglobulin. V. Tryptic fragments of carp (Cyprinus carpio L.) immunoglobulin M]

Carp IgM, isolated from normal serum is more sensitive to trypsinization compared to a human myeloma protein IgMGo. Under the same conditions (treatment with trypsin at 56 degrees C for 30 min) carp IgM was degraded to small, mostly dialysable peptides to a larger extent than IgMGo. In both cases the fragmentation resulted in immunoelectrophoretically pure Fab mu and Fc mu fragments. The Fab mu fragments of human IgM (yield: 20% of used IgM material) had a molecular weight of 54,000, the Fc mu fragments (yield: 30%) were a heterogenous mixture as far as molecular sizes concerned with values of about 300,000. For the corresponding fragments of carp IgM we could analyze a molecular weight of about 43,000 for Fab mu (yield: 8%) and for Fc mu (yield 10%) three fractions of 160,000, 130,000 and 90,000. The reductive subunits of Fc mu fragments showed different molecular weights: 39,000 for IgMGo and 45,000 for carp IgM. The anti-fragment antisera prepared in rabbits were monospecific as demonstrated by immunodiffusion.     

Elevation of plasma cortisol during the spawning migration of landlocked kokanee salmon (Oncorhynchus nerka kennerlyi)

Kokanee salmon (Oncorhynchus nerka kennerlyi ), a landlocked subspecies of sockeye salmon, exhibited hypothalamic-pituitary interrenal (HPI, adrenal homologue) axis activation and an increase in plasma cortisol concentration up to 639 +/- 55.9 ng/ml in association with upstream migration in the upper Colorado River even though they were not exposed to a change in salinity and lengthy migration. Kokanee salmon were collected at various stages of migration and concomitant sexual maturation. The pattern of cortisol elevation in kokanee is similar to that in ocean-run sockeye salmon (O. nerka nerka). The presence of plasma cortisol elevation in an upstream migrating, landlocked Pacific salmon suggests that stressors previously considered to cause the cortisol increase, such as long-distance migration and changes in salinity, may not be primary causes of the HPI axis activation.     

饲料不同水平淀粉对全养殖周期异育银鲫“中科3号”生长和糖代谢的影响

研究探讨了饲料淀粉对全养殖周期异育银鲫(Carassius auratus gibelio)生长性能和糖代谢的影响。实验以玉米淀粉为主要糖源, 分别设计淀粉水平为3% (S3)、13% (S13)、23% (S23)、33% (S33)和43% (S43)的5种等氮等脂饲料, 在长江故道江面网箱中饲养初始体重为(12.0±0.1) g的异育银鲫幼鱼340d, 期间根据鱼体养成阶段共取样5次, 生长阶段分为63d(D63, 幼鱼期)、110d(D110, 养成前期)、223d(D223, 越冬期)、275d(D275, 越冬后)和340d(D340, 养成中后期)。研究结果表明, 幼鱼期D63异育银鲫后肠淀粉酶活力较低且对饲料淀粉水平变化不敏感, 自养成前期D110之后鱼体后肠淀粉酶活力增强且随饲料淀粉水平的变化产生波动。幼鱼期D63异育银鲫glut2 mRNA相对表达量无显著差异, 其他阶段异育银鲫glut2 mRNA相对表达量水平随饲料淀粉水平升高而升高。各个生长阶段异育银鲫糖酵解的敏感度高于糖异生代谢, 幼鱼期D63与养成前期D110异育银鲫糖脂转化能力较强。幼鱼期D63异育银鲫适宜饲料淀粉水平为23%; 养成前期D110异育银鲫适宜饲料淀粉水平减为13%; 越冬后D275组异育银鲫整体代谢旺盛, 43%淀粉组生长最好; 而在养成中后期D340异育银鲫饲料淀粉需求则再次降到33%。研究结果表明, 异育银鲫在养殖周期的不同阶段对饲料淀粉的需求和利用存在显著的差异, 可以为异育银鲫不同养殖阶段特有饲料配方的设计提供支撑。     

Characteristics of adenosine triphosphatase activity in carp erythrocytes treated with saponin

The activities of Ca-ATPase and Na,K-ATPase in saponin-treated erythrocytes of man, rat and carp were compared. It was shown that at free calcium concentrations lower than 1 microM the activity of Ca-ATPase in carp erythrocytes was by one order of magnitude lower than in rat erythrocytes and 3-4 times lower than in human red blood cells. At [Ca2+] = 0.4 microM the activities of Na,K-ATPase in all species under study were essentially the same. The increase in Ca2+ concentration up to 1 microM resulted in a 2-5-fold activation of Na,K-ATPase in rat and carp erythrocytes, respectively. In all cases studied a further elevation of free calcium concentration was accompanied by a decline of the Na,K-ATPase activity. It was shown that the Pi content in carp erythrocytes is 5-6 times as high as that in mammalian cells. This circumstance is a considerable obstacle to a detailed analysis of mechanisms of ATPase activity regulation in carp erythrocytes by methods used for determination of inorganic phosphate production.     

The influence of maternal condition on offspring performance in sockeye salmon Oncorhynchus nerka

Eggs were taken from adult sockeye salmon Oncorhynchus nerka that had reached their journey's end in spawn-ready and moribund condition, and fertilized by healthy males. Egg number, size, hatching success and offspring growth did not differ with maternal condition, which suggests the absence of any persisting physiological maternal effects. Differences were noted in the swimming behaviour and physiology of the offspring at parr stage. In a 30 min schooling test conducted using groups of five in a flume, parr from moribund females were more likely to fatigue, were not as tightly schooled, and had a diminished startle response, both in the per cent responding and the burst distance. In individual, confined swimming tests conducted within a tube, post-exercise plasma lactate concentration, which is an indicator of white muscle use, was greater for parr from moribund adult females. The moribund females also had elevated lactate following exercise (their migration), which suggests heritable differences may exist in muscle use. This study shows that juvenile O. nerka artificially propagated from females exhausted by their return migration can exhibit swimming performance differences, indicating that maternal condition may need to be considered in breeding programmes.