An Unusual Case of Gastric Gnathostomiasis Caused by Gnathostoma spinigerum Confirmed by Video Gastroscopy and Morphological and Molecular Identification.

Human gnathostomiasis is a harmful foodborne parasitic infection caused by nematodes of the genus Gnathostoma. Here, we report an unusual case of gastric gnathostomiasis seen in a hospital in Thailand along with the clinical characteristics, treatment, and outcome. A 39-year-old man presented with complaints of epigastric pain, dizziness, and history of passing dark, tarry stools for 2 days. The patient had a history of consuming raw freshwater fish. Supplementary differential diagnosis was performed via rapid serological testing, and presence of the causative agent was confirmed based on video gastroscopy, morphology of the removed parasite, and molecular identification. After its surgical removal from the stomach, the parasite was morphologically identified as Gnathostoma species. Molecular identification was performed via DNA extraction from the recovered worm, and amplification and sequencing of the second internal transcribed spacer (ITS2) region and partial cytochrome c oxidase subunit I (cox1) gene. The ITS2 and cox1 sequences were consistent with those of Gnathostoma spinigerum. Clinicians in endemic areas should therefore be aware of the rare clinical manifestations and use of supplementary serological tests to facilitate early diagnosis and treatment of gastric gnathostomiasis.

Larval Gnathostomes and Spargana in Chinese Edible Frogs, Hoplobatrachus rugulosus, from Myanmar: Potential Risk of Human Infection.

Chinese edible frogs, Hoplobatrachus rugulosus, were examined to estimate the potential risks of human gnathostomiasis and sparganosis in Myanmar. A total of 20 frogs were purchased in a local market of Yangon and examined with naked eyes and the artificial digestion method after skin peeling in June 2018 and June 2019. Larvae of gnathostomes and Spirometra (=spargana) were detected in 15 (75.0%) and 15 (75.0%) frogs with average intensities of 10.5 and 6.3 larvae per infected frog, respectively. Gnathostome larvae were 2.75-3.80 (av. 3.30) mm long and 0.29-0.36 (0.33) mm wide. They had a characteristic head bulb with 4 rows of hooklets, a muscular long esophagus, and 2 pairs of cervical sac. The mean number of hooklets were 41, 44, 47, and 50 on the 1st, 2nd, 3rd, and 4th row, respectively. Collected spargana were actively moving, particularly with the scolex part, and have ivory-white color and variable in size. Conclusively, it has been first confirmed that Chinese edible frogs, H. rugulosus, are highly infected with larval gnathostomes and spargana in this study. Consuming these frogs is considered a potential risk of human gnathostomiasis and sparganosis in Myanmar.

Morphological and molecular characteristics of adult worms of Gnathostoma Owen, 1836 (Nematoda) collected from domestic pigs in Dien Bien Province, northern Vietnam.

Gnathostomes are of interest because of their unique appearance and medical importance. Among 13 valid species of the genus Gnathostoma Owen, 1836 (Nematoda: Spirurida), two species, G. doloresi Tubangui, 1925 and G. hispidum Fedtschenko, 1872, are parasites of pigs but their larvae can infect humans to cause gnathostomiasis. In this study, we collected adults of Gnathostoma sp. from the stomach of domestic pigs (Sus scrofa domesticus Linnaeus) from Dien Bien Province, northern Vietnam. Morphologically, nematodes found here are most similar to G. doloresi with a slight difference in the spicules of males. In contrast, they are genetically distinct from G. doloresi and other species of Gnathostoma in both ITS2 and cox1 sequences. The findings of the present study suggest that specimens of Gnathostoma sp. found in Dien Bien are likely a new species and emphasise the need of further studies on the taxonomy and phylogenetic relationship of species of Gnathostoma. Special attention should also be paid to swine and human gnathostomiasis in Dien Bien Province, Vietnam and the neighbouring areas of China and Laos.

Cross-Reactivity Pattern of Asian and American Human Gnathostomiasis in Western Blot Assays Using Crude Antigens Prepared from Gnathostoma spinigerum and Gnathostoma binucleatum Third-Stage Larvae.

Gnathostomiasis is a zoonotic parasitosis endemic in many Asian and some Latin American countries. Most human infections are caused by Gnathostoma spinigerum in Asia and Gnathostoma binucleatum in the Americas, and recently, imported cases have been increasing among travelers returning from endemic regions. Confirmation of the clinical diagnosis relies largely on serologic tests, with a G. spinigerum-antigen-based immunoblot currently being the diagnostic method of choice. However, we repeatedly experienced that sera from patients with clinically suspected American gnathostomiasis gave negative results in this assay. Therefore, we used homologous methods to prepare G. spinigerum- and G. binucleatum-antigen-based immunoblot assays, and evaluated the cross-reactivity of the two assays. The results show incomplete cross-reactivity between the two assays: the G. spinigerum-antigen-based immunoblot apparently only detects Asian gnathostomiasis caused by G. spinigerum, whereas the G. binucleatum-antigen-based immunoblot is apparently capable of detecting American as well as Asian gnathostomiasis.

Larval Gnathostoma spinigerum Detected in Asian Swamp Eels, Monopterus albus, Purchased from a Local Market in Yangon, Myanmar.

The present study was performed to determine the infection status of swamp eels with Gnathostoma sp. larvae in Myanmar. We purchased total 37 Asian swamp eels, Monopterus albus, from a local market in Yangon in June and December 2013 and 2014. All collected eels were transferred with ice to our laboratory and each of them was examined by the artificial digestion technique. A total of 401 larval gnathostomes (1-96 larvae/eel) were detected in 33 (89.2%) swamp eels. Most of the larvae (n=383; 95.5%) were found in the muscle. The remaining 18 larvae were detected in the viscera. The advanced third-stage larvae (AdL3) were 2.3-4.4 mm long and 0.25-0.425 mm wide. The characteristic head bulb (0.093 × 0.221 mm in average size) with 4 rows of hooklets, muscular long esophagus (1.025 mm), and 2 pairs of cervical sacs (0.574 mm) were observed by light microscopy. The average number of hooklets in the 1st, 2nd, 3rd, and 4th rows was 41, 45, 48, and 51, respectively. As scanning electron microscopic findings, the characteristic 4-5 rows of hooklets on the head bulb, a cervical papilla, tegumental spines regularly arranged in the transverse striations, and an anus were well observed. Based on these morphological characters, they were identified as the AdL3 of Gnathostoma spinigerum. By the present study, it has been confirmed for the first time that Asian swamp eels, M. albus, from Yangon, Myanmar are heavily infected with G. spinigerum larvae.

The Jeju weasel, Mustela sibilica quelpartis, a new definitive host for Gnathostoma nipponicum Yamaguti, 1941.

Adult gnathostomes were discovered in the stomach of the Jeju weasel, Mustela sibilica quelpartis, road-killed in Jeju-do (Province). Their morphological characters were examined to identify the species. Total 50 gnathostome adults were collected from 6 out of 10 weasels examined. In infected weasels, 4-6 worms were grouped and embedded in each granulomatous gastric tumor, except 1 weasel. Male worms were 25.0×1.4 mm in average size, and had a tail with pedunculate papillae, a spicule, and minute tegumental spines. Females were 40.0×2.5 mm in average size, and had a tail without tegumental spines. Pointed and posteriorly curved hooklets were arranged in 8-10 rows on the head bulb. Tegumental spines were distributed from behind the head bulb to the middle portion of the body. The spines were different in size and shape by the distribution level of the body surface. Fertilized eggs were 65.5×38.9 µm in average size, and had a mucoid plug at 1 pole. These gnathostomes from Jeju weasels were identified as Gnathostoma nipponicum Yamaguti, 1941. By the present study, it was confirmed for the first time that G. nipponicum is distributed in Jeju-do, the Republic of Korea, and the Jeju weasel, M. sibilica quelpartis, plays a crucial role for its definitive host.

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.

Is Gnathostoma turgidum an annual parasite of opossums? Drastic seasonal changes of infection in Didelphis virginiana in Mexico.

Gnathostoma turgidum is a nematode that parasitizes the stomach of opossums, Didelphis virginiana. Despite its wide distribution in the Americas, its natural life cycle is poorly understood. Recently, we found an endemic area for G. turgidum infection in Sinaloa, Mexico (Diaz-Camacho et al., 2009). Based on sporadic surveys for several years, the prevalence was apparently high in summer and extremely low in winter. To confirm that this is really a seasonal variance, we conducted a longitudinal survey on G. turgidum infection in opossums from November 2007 to November 2008. The results showed amazing seasonal changes in the prevalence, with synchronized migration and maturation of worms in opossums. Between February and March, many juvenile worms, with occasional AL3, were found in the liver, but no worms were found in the stomach. Mature adult worms began to appear in the stomach around April and rapidly increased in number toward July, when all worms resided in the stomach. Then, the worms disappeared almost completely by November. These results suggest that G. turgidum is an annual parasite of the opossum, D. virginiana, in Mexico.

Finding advanced third-stage larvae of Gnathostoma turgidum Stossich, 1902 in Mexico from natural and experimental host and contributions to the life cycle description.

In order to clarify the role of Gnathostoma turgidum as an etiological agent involved in human gnathostomiasis in Mexico, establish the taxonomic identity of the advanced third-stage larvae (AdvL(3)), and contribute to the knowledge of its life cycle, experimental host infections, examination of potential natural hosts, and morphological comparisons were carried out. Examination of ten species of potential hosts at San Pedro las Playas and Tres Palos Lagoon in Guerrero state, Mexico revealed that two (Kinosternon integrum and Rana zweifeli) were infected by 15 AdvL(3) of G. turgidum. A specific identity was obtained comparing these larvae with those recovered from hosts experimentally infected. The AdvL(3) measured 1.6 mm in length, with two cervical papillae (both in 12th row) and an excretory pore on the 19th row. The average of cephalic hooklets, from first to fourth row, was 30.8, 34.0, 36.7, and 39.6, respectively. This is the first record of AdvL(3) of G. turgidum in America, and it represents a significant contribution for the understanding of the life cycle of this species.

Abnormalities on cephalic hooklets of advanced third-stage larvae from Gnathostoma Owen, 1836 (Nematoda: Gnathostomidae) collected from Mexican rivulus Millerichthys robustus Costa, 1995 (Ciprinodontiformes: Rivulidae) in Tlacotalpan, Veracruz, Mexico.

Morphological abnormalities were observed on the cephalic bulb hooklets of advanced third-stage larvae (AdvL(3)) of genus Gnathostoma. The larvae were obtained from the fish "Mexican rivulus" Millerichthys robustus collected from a seasonal pond near Tlacotalpan, Veracruz, Mexico. The abnormalities involved (1) extra rudimentary hooklets, located between the four rows and after the fourth row, (2) branched or lobulated hooklets, and (3) fragmented hooklets not uniformly disposed in rows. The alterations observed on the cephalic bulb hooklets do not represent intraspecific variations, and they may be considered as a potential tool for assessing the presence of pollutants or stressors located within the ecosystem.

[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.

Confirmation of Gnathostoma binucleatum Almeyda-Artigas, 1991, advanced third-stage larvae in Tres Palos Lagoon, Mexico, by morphological and molecular data.

Advanced third-stage larvae of Gnathostoma sp. corresponding to 6 morphotypes, distinguished on the basis of the shape of the cephalic hooklets, were collected from the muscle tissue of 5 species of freshwater fish (i.e., Dormitator latifrons, Eleotris picta, Gobiomorus maculatus, Ariopsis guatemalensis, and Cichlasoma trimaculatum) in Tres Palos Lagoon, Guerrero, Mexico. Principal components analysis of 4 morphological characters cluster all samples in a single taxonomic group. A minimum amount of variation was observed among ITS2 sequences of 3 morphotypes and Gnathostoma binucleatum obtained from GenBank (0-0.84%). The observed variation among morphotypes 1, 2, and 3 is the result of intraspecific variability of G. binucleatum supported by morphology and DNA. Morphotypes 4, 5, and 6 belong to the same taxon on the basis of morphology of the hooklets only. For an accurate morphological diagnosis of the causative agent of gnathostomiasis, it is necessary to develop similar studies with other species of the genus.

New characters in the Gnathostomulid mouth parts revealed by scanning electron microscopy.

An analysis with SEM of the mouth parts of 16 species belonging to 10 genera of Gnathostomulida resulted in the following new characters and conclusions: 1) At least in the genus Haplognathia, jaw teeth that are visible by conventional light microscopy are composed of the same aggregated needle-like denticles that are found, often in large numbers, on the basal plates of many filospermoid species. 2) Other new ultrastructural tooth features include the tricuspid basal plate teeth in Problognathia minima, tripartite teeth in Austrognathia and Austrognatharia, and the clear separation, in the Gnathostomula basal plate, of a mediodorsal set of teeth from a more extensive rostroventral set. 3) Three rows of teeth, as typical for Gnathostomulidae and Austrognathiidae, are also present in the filospermoid Haplognathia filum. 4) The wide range of geographic variation in Haplognathia ruberrima is confirmed by significant differences in jaw teeth between specimens from Belize and Bermuda. 5) A compartmentalized involucrum is present in Labidgonathia longicollis. 6) A pair of lamellae addentales, until now only known from Valvognathia pogonostoma, was found in Tenuignathia rikerae, Problognathia minima, and probably also Labidognathia rikerae. 7) In all gnathostomulids, the lamella symphysis is composed of identical rods that are considered homologous with those in the mouth parts of Rotifera and Micrognathozoa.

Various morphologic features of Gnathostoma spinigerum in histologic sections: report of 3 cases with reference to topographic study of the reference worm.

Gnathostomiasis is common in Southeast Asian countries and can be found sporadically in other parts of the world mainly due to human migration. The definitive diagnosis can be given either by identification of the parasite isolated from the patient or through histologic section of the lesion. It is therefore important for pathologists to be familiar with the morphology of parasitic larvae which varies according to the levels of section-cutting so that the diagnosis will not be misled. We present three cases of gnathostomiasis with different features of parasitic morphology and compare these with the reference adult worm.

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.

Micrognathozoa: a new class with complicated jaws like those of Rotifera and Gnathostomulida.

A new microscopic aschelminth-like animal, Limnognathia maerski nov. gen. et sp., is described from a cold spring at Disko Island, West Greenland, and assigned to Micrognathozoa nov. class. It has a complex of jaws in its pharynx, and the ultrastructure of the main jaws is similar to that of the jaws of advanced scleroperalian gnathostomulids. However, other jaw elements appear also to have characteristics of the trophi of Rotifera. Jaw-like structures are found in other protostome taxa as well-for instance, in proboscises of kalyptorhynch platyhelminths, in dorvilleid polychaetes and aplacophoran mollusks-but studies of their ultrastructure show that none of these jaws is homologous with jaws found in Gnathostomulida, Rotifera, and Micrognathozoa. The latter three groups have recently been joined into the monophylum Gnathifera Ahlrichs, 1995, an interpretation supported by the presence of jaw elements with cuticular rods with osmiophilic cores in all three groups. Such tubular structures are found in the fulcrum of all Rotifera and in several cuticular sclerites of both Gnathostomulida and Micrognathozoa. The gross morphology of the pharyngeal apparatus is similar in the three groups. It consists of a ventral pharyngeal bulb and a dorsal pharyngeal lumen. The absence of pharyngeal ciliation cannot be used as an autapomorphy in the ground pattern of the Gnathifera because the Micrognathozoa has the plesiomorphic alternative with a ciliated pharyngeal epithelium. The body of Limnognathia maerski nov. gen. et sp. consists of a head, thorax, and abdomen. The dorsal and lateral epidermis have plates formed by an intracellular matrix, as in Rotifera and Acanthocephala; however, the epidermis is not syncytial. The ventral epidermis lacks internal plates, but has a cuticular oral plate without ciliary structures. Two ventral rows of multiciliated cells form a locomotory organ. These ciliated cells resemble the ciliophores present in some interstitial annelids. An adhesive ciliated pad is located ventrally close to a caudal plate. As in many marine interstitial animals-e.g., gnathostomulids, gastrotrichs, and polychaetes-a special form of tactile bristles or sensoria is found on the body. Two pairs of protonephridia with unicellular terminal cells are found in the trunk; this unicellular condition may be the plesiomorphic condition in Bilateria. Only specimens with the female reproductive system have been found, indicating that all adult animals are parthenogenetic females. We suggest that 1) jaws of Gnathostomulida, Rotifera, and the new taxon, Micrognathozoa, are homologous structures; 2) Rotifera (including Acanthocephala) and the new group might be sister groups, while Gnathostomulida could be the sister-group to this assemblage; and 3) the similarities to certain gastrotrichs and interstitial polychaetes are convergent.

A survey of infective larvae of Gnathostoma in eels sold in Ho Chi Minh City.

To investigate the distribution of Gnathostoma spp in Ho Chi Minh City (HCM city), 1,081 eels were purchased from a local market twice a month from March 1998 to February 1999. Infective larvae of Gnathostoma spp detected from the flesh and liver of eels by the press preparation technique were examined and identified. Three hundred and fifty advanced third-stage larvae were recovered from liver, none from the flesh. The average rate of infection was 0.11; a high rate of infection was found from August to November and a low rate of infection from February to May. The average number of larvae/eel was 2.9; the greatest number of larvae/eel was in January whereas the lowest was in March and April. There was a marked decrease in both prevalence and intensity of infection from February to May, followed by a rise from June. The finding suggests that in HCM city, the infection rate abruptly decreases soon after the end of the rainy season and starts to rise when the rain comes and reaches its peak at the end of the rainy season. All recovered larvae were identified as G. spinigerum.

Morphological variation and abnormality of cephalic hooklets of Gnathostoma spinigerum hepatic stage larvae from laboratory infected mice.

One thousand advanced third-stage larvae of Gnathostoma spinigerum from laboratory infected mice, two to five weeks after being fed with infected cyclops, were examined specifically for the morphology of their cephalic hooklets. Among these, only the 15-day old (early hepatic-stage) larvae and the 30-day old (late hepatic-stage) larvae were measured for the size of their body and hooklets. The average body size of the 15-day old and 30-day old larvae were 3.4 +/- 0.4 x 0.4 +/- 0.04 mm and 4.9 +/- 0.4 x 0.5 +/- 0.04 mm, respectively. The average size of the hooklets from rows one to four of the 15-day old larvae was 14.6 +/- 1.7 x 6.8 +/- 0.6 microm, 15.6 +/- 2.0 x 7.2 +/- 0.5 microm, 16.0 +/- 1.8 x 7.4 +/- 0.6 microm and 15.9 +/- 1.9 x 7.3 +/- 0.6 microm, respectively. Those of the 30-day old larvae were 15.1 +/- 1.7 x 7.1 +/- 0.6 microm, 16.3 +/- 1.6 x 7.5 +/- 0.7 microm, 16.5 +/- 1.7 x 7.8 +/- 0.6 microm and 16.3 +/- 1.7 x 7.6 +/- 0.8 microm, respectively. The average number of cephalic hooklets from rows one to four of the two- to five-week old larvae were 42.8 +/- 2.6, 45.3 +/- 2.8, 46.9 +/- 2.8 and 50.2 +/- 2.9, respectively. Several types of morphological variation and abnormality of the cephalic hooklets were observed. The most common ones were extra rudimentary hooklets below row four or within the four rows of hooklets (10.8%), the present of a fifth row of hooklets (1.9%), abnormal hooklets in only row four (1.2%), lobed or branched hooklets (0.5%), spiral arrangement of the four rows of hooklets (0.4%), and fragmented hooklets (0.4%).