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.

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

La gnatostomiasis es una zoonosis parasitaria causada por algunas especies del género de helmintos Gnathostoma . La presentación clínica es muy variada y su diagnóstico constituye un desafío; es endémica en países tropicales y subtropicales, y la transmisión se asocia con el consumo de carne de animales acuáticos, cruda o mal cocida. En las últimas décadas, el creciente turismo internacional y el consumo de alimentos exóticos, han provocado un notable aumento de casos de la enfermedad. En nuestro país no se ha reportado ningún caso confirmado de gnatostomiasis. Se presenta el caso de un hombre colombiano de 63 años de edad, viajero frecuente al exterior, que consultó por presentar síntomas gastrointestinales. Durante la hospitalización desarrolló una lesión cutánea en el flanco abdominal derecho, de la cual se extrajo una larva cuya descripción morfológica permitió clasificarla como Gnathostoma spinigerum . Se trata de la primera confirmación de un caso importado de gnatostomiasis en Colombia. En este artículo se discuten las generalidades, la etiología, la patogenia y el tratamiento de la enfermedad, con especial énfasis en las características particulares de este paciente.

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.

[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.

Growth and development of Gnathostoma spinigerum (Nematoda: Gnathostomatidae) larvae in Mesocyclops aspericornis (Cyclopoida: Cyclopidae).

BACKGROUND: Gnathostoma spinigerum larva is pathogenic, causing gnathostomiasis in humans and certain animals, and is prevalent mainly in Asia. Growth and development of Gnathostoma spinigerum larvae in the cyclopoid copepod Mesocyclops aspericornis, the first intermediate host, were examined. RESULTS: When newly hatched, ensheathed second-stage larvae (L2) were ingested by M. aspericornis, they immediately appeared exsheathed in the stomach of M. aspericornis. They then penetrated the stomach wall and entered the body cavity, where they immediately metamorphosed to a stunted form with the body length/width ratio equal to the early third-stage larvae (EL3) up to 2 h after being ingested. During metamorphosis, the anterior spine-like structure of L2 transformed into unequal transparent lips. The larvae moulted into EL3 in the body cavity of the copepod at around day 5-7 post-infection. Minute cuticular striations were seen on the whole body, with prominent single-pointed spines on the anterior part of the body. The head bulb had four rows of hooklets and two lateral trilobed lips. The size of EL3 in copepods continuously increased towards day 12 and showed a negative correlation to their density per copepod (R = -0.881, P < 0.05 for body length, and R = -0.906, P < 0.05 for body width). CONCLUSIONS: The results revealed for the first time that M. aspericornis, one of the most abundant freshwater copepods in Thailand, is a suitable first intermediate host for G. spinigerum. High susceptibility of M. aspericornis suggests its importance for the maintenance of the life cycle of G. spinigerum in Thailand.

Effect of some physical factors on the viability of third-stage Gnathostoma binucleatum larvae.

To diminish the risk of transmission to humans of advanced third-stage larvae (A3L) of Gnathostoma binucleatum in fish foods, we evaluated the effects of some physical factors on larval viability. A3L protected within fish meatballs were subjected to freezing, refrigeration, boiling, dry heat, and immersion in lemon juice. By freezing, larvae were killed in 48 h, by refrigeration after 30 days, by boiling in 4 min, and by broiling for 60 min. By lemon juice immersion (pH 2.5), encysted larvae were killed after 5 days and nonencysted larvae in 7 h. Results show that freezing fish at -10 to -20°C for 48 h, or cooking fish by frying, boiling, or broiling, will prevent transmission of G. binucleatum. Furthermore, results dispel the popular myth that lemon juice kills encysted larvae in fish.

Intrahepatic growth and maturation of Gnathostoma turgidum in the natural definitive opossum host, Didelphis virginiana.

Gnathostoma turgidum is a gastric nematode parasite of opossums found in the Americas. We recently found that G. turgidum juveniles appear in the liver of the opossums where they become mature adults and almost synchronously move to the stomach during certain months of the year, suggesting the importance of the liver for the growth and maturation of this species in the final hosts. In this study we attempted to detect G. turgidum larvae in the liver of opossums, Didelphis virginiana that are the natural final hosts. The results show that tiny (<3mm in length) third stage larvae (L3) appeared in the liver of opossums around November and December. Also in the liver, we found large L3 of up to about 10mm in length together with juveniles and mature adults from February to March. In spite of their length, large L3 have 4 rows of hooklets, and their gonads remained undeveloped. Morphological features of the small and large L3 of G. turgidum are described including scanning electron microscope images. The seasonal switching of the several growth stages of G. turgidum from small L3 to adult worms in the liver and eventual migration to the stomach in opossums suggests the unique feature of G. turgidum utilizing the liver as the maturation site.

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.

Molecular identification of the advanced third-stage larvae (ADV L(3)) of Gnathostoma lamothei in Tabasco, Mexico.

Advanced third-stage larvae (ADV L(3)) of Gnathostoma spp. were collected from the muscle tissue of three species of freshwater fish (i.e., Gobiomorus dormitor, Petenia splendida, and Parachromis managuensis) in Swamps of Centla, Tabasco, Mexico. Nine sequences of the ITS2 of the ribosomal DNA of Gnathostoma spp. were compared with sequences obtained from GenBank for G. binucleatum, G. lamothei, G. miyazakii, G. spinigerum, and G. turgidum. Sequences of the ADV L(3) from P. splendida (Isla Chinal), P. managuensis (Isla Chinal), and of two of the six larvae collected from G. dormitor (Tres Brazos), were identical to that of G. binucleatum (GenBank). Sequences from the other four larvae from G. dormitor (Tres Brazos) are identical to the sequence of G. lamothei (GenBank). This is the first record of the intermediate host of G. lamothei. The only species documented to cause human gnathostomiasis in the Americas is G. binucleatum. Our finding of G. binucleatum, and G. lamothei parasitizing the commercially important fish species, G. dormitor in Centla swamps, indicates the possibility of G. lamothei causing human gnathostomiasis in Mexico as well.

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.

Short report: case of gnathostomiasis in Beijing, China.

A 59-year-old woman sought treatment of for creeping eruption and erythematous plaques associated with high fever and systemic symptoms. She had a history of eating undercooked freshwater fish raised in Beijing 10 days before admission and reported no travel during the previous year. Blood examination showed eosinophila and ultrasonography detected multiple hypoechoic areas in the liver and spleen. Western blot test detected specific antibodies to the larvae of Gnathostoma spinigerum. Cutaneous and visceral larva migrations associated with G. spinigerum were diagnosed. Twenty-three cases in 12 provinces have been reported in the Chinese literature but none have been reported in English.

[Gnathostoma (Spirurida: Gnathostomatidae) infection in the tigerfish Hoplias microlepis: prevalence, correlation with fish size, hosts, and public health implications]. / Infección por Gnathostoma (Spirurida: Gnathostomatidae) en Hoplias microlepis: prevalencia, correlación con la talla del pez, huéspedes e implicaciones para salud pública en Ecuador.

INTRODUCTION: Human gnathostomiasis has been reported in Ecuador since the early 1980s, when natural infections by Gnathostoma third larval stages were found in muscles of the second intermediary host, Hoplias microlepis (tigerfish). In Ecuador, this zoonotic disease is occasionally detected in humans, and its monitoring and eco-epidemiological assessment is of particular interest for its detection and control. OBJECTIVE: The most recent evidence is provided with respect to natural infections by Gnathostoma as it occurs in the tigerfish (Hoplias microlepis), including insights into its biological cycle. MATERIALS AND METHODS: A total of 74 fish were collected from two localities (rice fields-wetlands and local fish market) of Samborondón County (Guayas Province). Each was examined for the presence of Gnathostoma in muscle of Hoplias microlepis. The abundance intensity and prevalence of parasites was estimated. Statistical comparisons between the two sites and correlations of parasite load versus fish size were conducted. RESULTS: The infection prevalence by Gnathostoma was 69% (95% CI: 57-78%). The overall abundance intensity of parasites averaging the 2 sites was 1.7 larvae per fish. The proportion of infected fish was higher in rice fields (77%) when compared to those from the local fish market (62%). No statistically significant differences between the abundance intensity and prevalence were found between the sites (p>0.05). Parasite load and fish length were significantly correlated (Spearman p<0.05). CONCLUSIONS: Gnathostomiasis is a prevalent zoonosis in coastal Ecuador, and its etiologic agent is commonly found in the second intermediary host. Several Neotropical mammals are candidates as definitive reservoir hosts in the Gnathostoma biological cycle.

[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.

[Gnathostomosis, an exotic disease increasingly imported into Western countries]. / La gnathostomose, une maladie exotique de plus en plus souvent importée dans les pays occidentaux.

AN INCIDENTAL HELMITHIASIS IN MAN: Gnathostomiasis is an helminthic disease of animals due to a nematode belonging to the gender Gnathostoma. This gender includes many species, the most frequent being Gnathostoma spinigerum. Man is an incidental host. Human gnathostomiasis is endemic in some countries of South-East Asia, and Latin America. It is due to the consumption of raw or insufficiently cooked meat or fish. Since the beginning of the eighties, there is an increasing number of cases of gnathostomiasis described in Western countries in travellers returning from endemic countries. IN THE SKIN OR THE VISCERA: Gnathostomiasis is a cause of cutaneous and/or visceral larva migrans syndrome. Some visceral involvement, more particularly neurological forms, may lead to significant morbidity and mortality. The diagnosis is occasionally confirmed by the identification of the Gnathostoma larva in the skin or viscera. Most often the diagnosis relies on epidemiological, clinical and biological (hypereosinophilia, positive serologic test) grounds. ALBENDAZOLE AND IVERMECTINE: The first line treatment is albendazole, 400 mg once or twice a day during 21 days. The efficacy of ivermectin needs to be assessed more precisely. Relapses may occur up to 24 months after apparent cure.

A note on the correlation between rainfall and the prevalence of Gnathostoma spp. infective stage larvae in swamp eels in Thailand.

Gnathostoma spinigerum is the major causative agent of human gnathostomiasis, a parasitic zoonosis with a great variety of clinical manifestations. Generally, humans are infected by consumption of third-stage larvae (L3) of G. spinigerum in infected hosts in the form of partially cooked or uncooked food. Surveys of the contamination of Gnathostoma spp. L3 in swamp eels are useful for prevention and control of diseases and have been continuously performed in Thailand. The author performed a retrospective study on 33 previous cross-sectional surveys with geographical data and the prevalence of Gnathostoma spp. L3 that covered 12 provinces in Thailand. The relation between rainfall (derived from the geographical data) and the prevalence of Gnathostomo spp. L3 in swamp eels (derived from the overall infection rate of Gnathostoma spp. L3) was investigated. The least-square equation plot rainfall (y) versus prevalence (x) is y= 9.68x + 1,035.12 (r = 0.83; p < 0.01). A significant correlation was discerned between rainfall and the prevalence of eel infection but not for the season of the survey. Similar to the previous study, the prevalence of eel infection may depend on rainfall rather than season. However, this study focused on only 33 cross-sectional surveys in Thailand; further similar study in other countries to assess the correlation between rainfall and the prevalence of infection is required to substantiate this conclusion.

Study of specific IgG subclass antibodies for diagnosis of Gnathostoma spinigerum.

Gnathostoma spinigerum infection is endemic in Thailand and many Asian countries. Current diagnosis is the skin test and enzyme-linked immunosorbent assay (ELISA) for IgG antibody against the G. spinigerum third-stage larvae (L3), but cross-reactivity is common. We evaluated the sensitivity and specificity of anti-G. spinigerum L3 IgG subclass antibodies for diagnosis of 43 patients with gnathostomiasis. The majority of patients with gnathostomiasis (91%) had eosinophilia. While the anti-G. spinigerum L3 IgG1 antibody provided the highest sensitivity (98%), the anti-G. spinigerum L3 IgG2 antibody had the highest specificity (88%). The ELISA that detected anti-G. spinigerum L3 IgG1 antibody could be a reliable laboratory screening test, while anti-G. spinigerum L3 IgG2 antibody could be used to confirm the diagnosis.