Effects of Gnathostoma spinigerum infective stage larva excretory-secretory products on NK cells in peripheral blood mononuclear cell culture: focused on expressions of IFN-γ and killer cell lectin-like receptors.

Human gnathostomiasis is mainly caused by third-stage larvae of Gnathostoma spinigerum (G. spinigerum L3). Excretory-secretory products (ES) released from infective helminthic larvae are associated with larval migration and host immunity modulation. Natural killer (NK) cells have important immune functions against helminth infection. Currently, the effects of ES from G. spinigerum L3 (G. spinigerum ES) on NK cell activity are unclear. This study investigated whether G. spinigerum ES affected human NK cells. Human normal peripheral blood mononuclear cell (PBMC) cultures were used to mimic immune cells within the circulation. PBMC were co-cultured with G. spinigerum ES (0.01-0.05 µg/ml) for 5 or 7 days. Levels of IFN-γ in cultured supernatants were measured by enzyme-linked immunosorbent assay. The expressions of mRNA encoding NK cell receptors, especially the C type killer cell lectin-like family (KLR; NKG2A, NKG2C, and NKG2D) and IFN-γ in ES induced PBMC were determined by quantitative reverse transcription-polymerase chain reaction (RT-qPCR). ES induced PBMC markedly decreased the levels of IFN-γ and increased the expressions of NKG2A and NKG2D on NK cells. In conclusion, low amounts of G. spinigerum ES modulated NK cells by downregulating the transcription of IFN-γ and upregulating the expressions of KLR (NKG2A and NKG2D receptors) during the 7-day observation period. These findings indicate more in-depth studies of NK cell function are required to better understand the mechanism involved in immune evasive strategies of human gnathostomiasis.

Excretory-secretory product of third-stage Gnathostoma spinigerum larvae induces apoptosis in human peripheral blood mononuclear cells.

Human gnathostomiasis caused by third-stage Gnathostoma spinigerum larvae (G. spinigerum L3) is an important zoonotic disease in tropical areas of the world. The excretory-secretory products (ES) that are excreted by infective larva play a significant role in host immune evasion and tissue destruction. To investigate the poorly understood mechanisms of G. spinigerum L3 pathogenesis, we focused on the potential effect of ES on inducing apoptosis in human immune cells by using human peripheral blood mononuclear cells (PBMCs) as a model. Early and late apoptosis of PBMCs were assessed following the exposure of these cells to G. spinigerum L3 ES (0.1, 0.5, and 1.0 µg/ml) for 6-48 h. The apoptotic cells were identified by flow cytometric staining of PBMC with FITC-annexin V and propidium iodide. The expression of regulatory genes related to apoptosis mechanisms in ES-treated PBMCs was investigated using a Human Apoptosis RT2 Profiler™ PCR Array. The results showed significant levels of early phase apoptosis at 18 h and of late phase apoptosis at 24 h. We speculate that this apoptosis in PBMCs occurs via the extrinsic pathway. Apoptosis in the ES-induced PBMCs was observed as quickly as 90 min after exposure, and the highest effect was observed at 18-24 h. Furthermore, ES can trigger apoptosis lasting for 48 h. Our findings expand the understanding of one of the mechanisms involved, immune-evasive strategy mechanism used by G. spinigerum larvae during human gnathostomiasis.

Description and development of the early third-stage larva of Gnathostoma turgidum Stossich, 1902 (Nematoda: Gnathostomatidae) and contributions to its life cycle.

The egg and larval stages of Gnathostoma turgidum were examined using light microscopy. Fertilized uterine eggs are 65.97 long and 32.28 wide, oval, brownish, with two cap-like thickenings. The eggshell surface is covered with numerous irregularly shaped pits of various sizes and depths. A sheathed second-stage larva emerges from the egg, measures 178 x 9; the sheath measures 243 x 21. Development to early third-stage larva in the coelomic cavity of cyclopoid copepods is similar to that described for other gnathostome species. After 10 days at 27 degrees C, the larvae undergo a molt (the second for gnathostomes) and develop to early third stage. The body of this stage measures 412.3 x 40.1, with evident hemispherical cephalic bulbs. Cephalic bulbs measure 25 x 40, armed with four transverse rows of sharp hooklets. The average number of hooklets in each row is 31, 34, 37, and 42, respectively. The whole body is covered with 193 transverse rows of small single-pointed cuticular spines. One pair of cervical papillae and an excretory pore are present on the anterior part of the body. On the other hand, potential species-specific features regarding the latter larval stage are discussed. Finally, some G. turgidum life cycle considerations are portrayed.

Precocity of Gnathostoma turgidum in naturally infected four-eyed opossum Philander opossum pallidus from Temascal, Oaxaca, Mexico.

Two female advanced third-stage larvae of Gnathostoma turgidum recovered from the liver of one naturally infected four-eyed opossum Philander opossum pallidus collected in Oaxaca, Mexico, were morphologically examined. Because of some characteristics, the larvae do not fit into the typical advanced third-stage. The body shows a size at least three times larger than expected and rows of spines only in the anterior part of the body surface. Consequently, in this research, we document for the first time the precocity in third-stage larvae of G. turgidum, and we also highlight some facts about the fourth larval stage occurring in spirurins.

Pila ampullacea and Pomacea canaliculata, as new paratenic hosts of Gnathostoma spinigerum.

Aquatic snails, Pila ampullacea and Pomacea canaliculata were experimentally found to be suitable paratenic hosts for advanced third-stage larvae (L3) of the nematode Gnathostoma spinigerum, the causative parasite of gnathostomiasis in humans. G. spinigerum (L3) were found to be encapsulated in the tissue of the snail's foot and its internal organs. The infection, intensity and survival of third-stage larvae of G. spinigerum in both species of aquatic snails are described. This is the first evidence to reveal that not only vertebrates but also invertebrates (snails) can serve as paratenic hosts to this parasite. Aquatic snails are one of several sources of human gnathostomiasis in Thailand.

[Potamotrygon marinae n. sp., a new species of freshwater stingrays from French Guiana (Myliobatiformes, Potamotrygonidae)]. / Potamotrygon marinae n. sp., une nouvelle espèce de raies d'eau douce de Guyane (Myliobatiformes, Potamotrygonidae).

Six specimens of freshwater stingrays from the French Guiana belonging to the genus Potamotrygon [S.W. Garman, On the pelvis and external sexual organs of selachians, with special reference to the new genera Potamotrygon and Disceus, Proc. Bost. Soc. nat. Hist. 19 (1877) 197-215], do not present characters that are typically shown by species to which they have been attributed. Five belong to a new species here named Potamotrygon marinae n. sp. This species is differentiated from the others by the feebly development of the prepelvic process, the development of the postorbital process as an enlarged blade, the unsegmented angular cartilage, the dorsal surface coloration composed of wide circular patches themselves formed by smaller pale patches, the almost dark coloration of the ventral surface tessellated with pale patches, and the small sized spiny tubercles situated in the middorsal region, before the caudal sting.

Gnathostomiasis: a review of a previously localized zoonosis now crossing numerous geographical boundaries.

Expansion in international travel and increases in immigration have resulted in an increased number of persons in Europe and other Western countries who have returned from foreign travel with parasitic infections rarely seen previously in the United States. Among the diseases caused by helminthic parasites is gnathostomiasis, a disease caused by Gnathostoma spp. Once confined primarily to Southeast Asia, it now is a public health concern in Mexico and other countries. This article reviews the causes and epidemiology of gnathostomiasis, the life cycle of the helminth, the clinical picture and diagnosis of gnathostomiasis, and the treatments for and means of avoiding the disease.

Analysis of sequence variation in Gnathostoma spinigerum mitochondrial DNA by single-strand conformation polymorphism analysis and DNA sequence.

Morphological variations were observed in the advance third stage larvae of Gnathostoma spinigerum collected from swamp eel (Fluta alba), the second intermediate host. Larvae with typical and three atypical types were chosen for partial cytochrome c oxidase subunit I (COI) gene sequence analysis. A 450 bp polymerase chain reaction product of the COI gene was amplified from mitochondrial DNA. The variations were analyzed by single-strand conformation polymorphism and DNA sequencing. The nucleotide variations of the COI gene in the four types of larvae indicated the presence of an intra-specific variation of mitochondrial DNA in the G. spinigerum population.

[Cutaneous gnathostomiasis, first confirmed case in Colombia]. / Gnatostomiasis cutánea, primer caso confirmado en Colombia.

Gnathostomiasis is a parasitic zoonosis caused by some species of helminthes belonging to the genus Gnathostoma . It has a wide clinical presentation and its diagnosis is a challenge. Tropical and subtropical countries are endemic, and its transmission is associated with eating raw or undercooked meat from fresh water animals. Increasing global tourism and consuming exotic foods have produced a noticeable rise in cases of the disease in the last decades. However, in our country, there has not been any confirmed case of gnathostomiasis previously reported. We present the case of a 63-year-old Colombian man with an international travel history, who presented with gastrointestinal symptoms. During the hospital stay, he developed a cutaneous lesion on the upper right abdominal quadrant, where later, a larva was found. A morphological study allowed us to identify it as Gnathostoma spinigerum . As such, this is the first report of an imported case of gnathostomiasis confirmed in Colombia. This article describes the principles, etiology, pathogenic cycle and treatment of this disease with special considerations to our patient´s particular features.

First reported case of intraocular Gnathostoma spinigerum in Cambodia.

A live intraocular nematode was identified from a 37 year-old man presented with iritis, pain, redness, lacrimation, swelling, vision loss and intermittent blindness during many hours per day of the left eye. By using slit lamp examination, a worm was removed from iris in an ophthalmology outpatient department setting and sent to the Medical Microbiology Laboratory, Institut Pasteur du Cambodge. Gnathostoma spinigerum was identified, based on its typical morphology via microscopic examination. Based on our diagnosis, the patient was treated by oral albendazole and responded well to this therapy.

Study of the ethiological agent of gnathostomosis in Nayarit, Mexico.

In order to clarify the specific identity of the etiological agent of human gnathostomosis in Nayarit State, Mexico, morphological and molecular studies were conducted on advanced third stage larvae obtained from human and fish tissue. Cathorops fuerthii from Agua Brava lagoons complex, was the only fish species found to be infected among four species surveyed. Morphological variability does not allow specific identification of the larvae. Internal transcribed spacer 2 of the ribosomal DNA was sequenced for six larvae (five from fish, one from human tissue). Low divergence in the sequences of Nayarit larvae and Gnathostoma binucleatum (0.24% or less) indicate that the larvae examined belong to this species.

Gnathostoma infective stage larvae in swamp eels (Fluta alba) at a metropolitan market in Bangkok, Thailand.

A total of 1,788 livers from swamp eels (Fluta alba), purchased at a market in Bangkok, Thailand, from April to July 2002, were studied for the prevalence and intensity of Gnasthostoma third stage larvae (L3s). Of all livers, 466 were infected with Gnasthostoma L3s (infection rate = 26%). The larvae recovered averaged 0.43 +/- 0.51 larvae/liver. All larvae were identified as Gnasthostoma spinigerm by morphological criteria. The prevalence of G. spinigerum L3s in the eels was much higher in the rainy season (June and July, 74%) than during April and May (8%). Therefore, prevention programs for gnathostomiasis should be emphasized during the rainy season.

The first record of a confirmed human case of Gnathostoma doloresi infection.

The first case of Gnathostoma doloresi infection in a human was found in Miyazaki Prefecture, Japan. A whole length of the parasite was dissected out from biopsied skin and was identified as the third-stage larva of G. doloresi based on the morphological characteristics of the hooklets of the head bulb and also on the number of nuclei in the intestinal epithelial cells of the parasite.

Gnathostoma spinigerum: growth and development of third-stage larvae in vitro.

Advanced third-stage larvae of Gnathostoma spinigerum were cultured in RPMI-1640, with various supplements at 37 C under 5% CO2 in air for 300 days. The most suitable medium supplement for worm development was 10% fetal calf serum, 1% dog serum, and 0.25% dog hemolysate. After approximately 180 days of cultivation, some larvae molted to the fourth stage as distinguished by 8 transverse rows of cephalic hooklets and well differentiated sex organs. The maximum body length and width of these larvae were 18.6 mm and 1.1 mm, respectively. Six of 50 larvae (12%) developed to the fourth stage, with a 32% survival rate at the end of cultivation. Although the highest survival rate (70%) of the worms was observed in the medium supplemented with 25 mM NaHCO3, only 4% developed into fourth stage larvae. The addition of fetal calf serum, dog serum, and dog hemolysate was essential for growth and development.

Molting of Gnathostoma doloresi (Nematoda: Gnathostomatoidea) in the definitive host.

Premolt, molting, and postmolt worms of Gnathostoma doloresi (Nematoda: Gnathostomatoidea) recovered from the stomach wall of naturally infected wild boars Sus scrofa leucomystax in Miyazaki, Japan, were examined morphologically. The only molt observed was that from the advanced third-stage to the adult stage. It is strongly suggested that the gnathostomes molt only once in the definitive host.

Susceptibility of several species of Cyprinidae and Salmonidae freshwater fish to larval Gnathostoma nipponicum infection.

Susceptibility of five species of Cyprinidae and Salmonidae freshwater fish to the early third-stage larvae (EaL3) and advanced third-stage larvae (AdL3) of Gnathostoma nipponicum infection were examined. Two fish species inoculated orally with EaL3 were infected, and AdL3 were recovered from them with rate of 21.0% in Tribolodon hakonensis and 0.5% in Cyprinus carpio at 30 days postinoculation (PI). Attempts to infect five fish species with AdL3 were all successful. The recovery rate of AdL3 was 69.0% in T. hakonensis, 47.5% in Carassius auratus subsp., 35.0% in C. carpio, 53.0% in Oncorhynchus masou, and 32.0% in O. mykiss at 10 days PI. These results confirmed that the Cyprinidae and Salmonidae fish species reported here were susceptible to larval G. nipponicum infection and AdL3 had higher infectivity to them than the EaL3.

Scanning electron microscopy of the early third-stage larvae of Gnathostoma spinigerum.

Scanning electron microscopic observations were made on the early third stage (eL3) larvae of Gnathostoma spinigerum (Sakolnakhon, northeast Thailand) from 3-week-old infected cyclops (Mesocyclops leuckarti). The morphological surfaces of the anterior end, head spine, body cuticle, amphid, papillae, posterior end of larvae were described and compared with the advance third-stage (aL3) larvae.

Prevalence and intensity of third stage Gnathostoma spinigerum larvae in swamp eels sold in three large markets in Bangkok, Thailand.

Gnathostoma spinigerum is a common human tissue parasite in Thailand. The swamp eel is the major intermediate or paratenic host for this parasite. The high prevalence of the infective third stage larvae (L3) of this parasite in the rainy season has been noted in previous studies. During June 1999 (rainy season), we performed a cross-sectional survey of the prevalence and the intensity of G. spinigerum L3s in the livers of swamp eels that were obtained from three large Bangkok markets (Klong Toey, Pran Nok, and Tevej). Of a total of 785 livers, G. spinigerum L3s were found in 97: an infection rate of 12%. The prevalence rates in Klong Toey, Pran Nok, and Tevej markets were 13%, 10% and 14% respectively. There was no significant difference in the proportion of infected eels between the markets (p > 0.05). The intensity of L3 burden in the livers ranged from 1-17 larvae, with an average of 2.60 +/- 0.24 larvae (mean +/- SE). In this study a high prevalence of G. spinigerum L3s was found; there was no significant difference in the intensity of larvae in swamp eels between markets. These findings emphasize the importance of public health education: people need to be aware of the dangers of consuming raw or undercooked food.

Comparative surface ultrastructure of adults and eggs of Gnathostoma obtained in Japan.

As limited studies have been done on surface morphology of Gnathostoma, adult specimens and eggs of four kinds of species in Japan were compared by scanning electron microscopy. Worms had a subglobular head-bulb which was armed with 7-10 rows of cephalic hooks. Mutidigitate cuticular spines were spaced unevenly on transverse cuticular striations on the anterior half of the body. The lengths of the spines were variable with tridentate spines longer than bidentate ones, These tridantate spines became one of the species specific characteristics. The posterior half of the bodies of G. doloresi and G. hispidum were covered densely with long unidentate spines which were gradually shorter towards the posterior ends. Ventral sides of male terminals had different shape of papillae which so called small and caudal ones in species. Eggs recovered from the uteri of female worms were covered with cuticular pits of different sizes, shapes and depths in species.

A survey of Gnathostoma larvae in fresh water fish in the valley of the Yangtze River and morphological characteristics of the recovered larvae.

Investigations of the prevalence of larval gnathostomes in fresh water fishes were carried out at the southeastern Yangtze Valley, People's Republic of China, in the periods of October 1989 and November 1990. Fishes were collected from Shanghai, Chenchiang, Nanching, Chiuchiang and Nanchang districts in 1989. Additional sampling in Shanghai district was done at Kunshan, Tien-shanfu, Chingpu and Nanhui. Species of fishes collected were Channa argus (110), Siniperca chuatsi (24) and Silurus asotus (2). Muscle tissue of the fishes was dissected into small pieces, sliced and then examined under a dissecting microscope. The viscera were pooled by species in groups of 4 or 5 individuals, homogenized, and were then digested overnight in artificial gastric-juice at 37 degrees C. Four encysted larvae were recovered from the muscle tissue of four C. argus. Thirty-four larvae were obtained from digestion of viscera. A total of 38 larvae were recovered. Eighteen of 38 larvae were examined morphologically and they were able to be divided into three types by their body length; 5 early third-stage larvae (0.58-0.86 mm), 12 third-stage larvae (1.12-2.61 mm), and one advanced third-stage larva of 4.86 mm. Light and scanning electron microscopy revealed that the former two types had characteristics of Gnathostoma hispidum and the last one had those of G. spinigerum. In 1990, we investigated fish near Hongtze-hu and Tai-hu lakes. A total of 553 fishes belonging to 12 genera and 12 species were examined. Seventeen larvae were recovered from the viscera of G. argus and Monopterus albus. These larvae were identified as G. hispidum.