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 Zoonotic Trematode Metacercariae in Fish from a Local Market in Yangon City, Myanmar.

A survey was performed to investigate the infection status of zoonotic helminth larvae in fish from a local market of North Dagon District in Yangon City, Myanmar. A total of 486 fish in 13 species were collected 8 times from December 2015 to December 2019. All fish were transported under ice to a laboratory in Korea and examined for helminth larvae using artificial digestion method. Larval gnathostomes and metacercariae of more than 8 zoonotic trematode species, i.e., Opisthorchis viverrini, Haplorchis taichui, H. pumilio, H. yokogawai, Centrocestus spp., Stellantchasmus falcatus, Pygidiopsis cambodiensis, and Procerovum sp., were detected. Larval gnathostomes were found in 58 (16.0%) out of 362 fish of 6 species, with mean intensity of 2.8 per fish infected. Metacercariae of O. viverrini were detected in 10 (2.9%) out of 349 fish of 5 species, with mean intensity of 16.9 per fish infected. Metacercarial prevalences of 4 intestinal flukes, H. taichui, H. pumilio, H. yokogawai, and Centrocestus spp., were 16.8%, 26.0%, 12.5%, and 15.0% in the positive fish species, respectively, and mean metacercarial intensity was 63.3, 26.8, 86.2, and 8.7 per fish infected. Metacercariae of S. falcatus and P. cambodiensis were detected only from the mullet, Chelon macrolepis. Metacercariae of Procerovum sp. were found in Channa striata and Anabas testudineus. Collectively, it was confirmed that the fish were infected with gnathostome larvae and metacercariae of O. viverrini and intestinal flukes in Yangon City, Myanmar.

Molecular Identification of the Etiological Agent of Human Gnathostomiasis in an Endemic Area of Mexico.

Human gnathostomiasis, which is endemic in Mexico, is a worldwide health concern. It is mainly caused by the consumption of raw or insufficiently cooked fish containing the advanced third-stage larvae (AL3A) of Gnathostoma species. The diagnosis of gnathostomiasis is based on epidemiological surveys and immunological diagnostic tests. When a larva is recovered, the species can be identified by molecular techniques. Polymerase chain reaction (PCR) amplification of the second internal transcription spacer (ITS-2) is useful to identify nematode species, including Gnathostoma species. This study aims to develop a duplex-PCR amplification method of the ITS-2 region to differentiate between the Gnathostoma binucleatum and G. turgidum parasites that coexist in the same endemic area, as well as to identify the Gnathostoma larvae recovered from the biopsies of two gnathostomiasis patients from Sinaloa, Mexico. The duplex PCR established based on the ITS-2 sequence showed that the length of the amplicons was 321 bp for G. binucleatum and 226 bp for G. turgidum. The amplicons from the AL3A of both patients were 321 bp. Furthermore, the length and composition of these amplicons were identical to those deposited in GenBank as G. binucleatum (accession No. JF919679), corroborating our previous morphological finding that G. binucleatum is the etiological agent for human gnathostomiasis in the endemic area of Sinaloa, Mexico.

Molecular identification and genetic diversity of Gnathostoma spinigerum larvae in freshwater fishes in southern Lao PDR, Cambodia, and Myanmar.

Gnathostomiasis, an emerging food-borne parasitic zoonosis in Asia, is mainly caused by Gnathostoma spinigerum (Nematoda: Gnathostomatidae). Consumption of raw meat or freshwater fishes in endemic areas is the major risk factor. Throughout Southeast Asia, including Thailand, Lao PDR, Cambodia, and Myanmar, freshwater fish are often consumed raw or undercooked. The risk of this practice for gnathostomiasis infection in Lao PDR, Cambodia, and Myanmar has never been evaluated. Here, we identified larvae of Gnathostoma species contaminating freshwater fishes sold at local markets in these three countries. Public health authorities should advise people living in, or travelling to, these areas to avoid eating raw or undercooked freshwater fishes. Identification of larvae was done using molecular methods: DNA was sequenced from Gnathostoma advanced third-stage larvae recovered from snakehead fishes (Channa striata) and freshwater swamp eels (Monopterus albus). Phylogenetic analysis of a portion of the mitochondrial cytochrome c oxidase subunit I gene showed that the G. spinigerum sequences recovered from southern Lao PDR, Cambodia, and Myanmar samples had high similarity to those of G. spinigerum from China. Sequences of the nuclear ribosomal DNA internal transcribed spacer 2 region closely resembled sequences of G. spinigerum from Thailand, Indonesia, the USA, and central Lao PDR. This is the first molecular evidence of G. spinigerum from freshwater fishes in southern Lao PDR, Cambodia, and Myanmar.

Genetic diversity of infective larvae of Gnathostoma spinigerum (Nematoda: Gnathostomatidae) in freshwater swamp eels from Thailand.

Human gnathostomiasis is a food-borne zoonosis caused by a tissue nematode of the genus Gnathostoma. The disease is highly endemic in Asia, including Thailand. The freshwater swamp eel (Monopterus albus), the second intermediate host of the gnathostome nematode, has an important role in transmitting the infection in Thailand. Surveys on the infective larvae of Gnathostoma spinigerum based on morphological features in freshwater swamp eels have been performed continuously and reported in Thailand. However, there is still limited molecular data on intra-species variations of the parasite. In this study, a total of 19 third-stage larvae of morphologically identified G. spinigerum were collected from 437 liver samples of freshwater swamp eels purchased from a large wholesale market in Bangkok, Thailand. Molecular characterization based on mitochondrial cytochrome c oxidase subunit I (COI) sequences was performed to elucidate their genetic variations and phylogenetic relationship. Among the 19 infective larvae recovered from these eels, 16 were sequenced successfully. Phylogenetic analyses inferred from the partial COI gene showed the presence of three distinct COI haplotypes. Our findings confirm the presence of G. spinigerum as the main species in Thailand.

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.

Gnathostoma spinigerum Mitochondrial Genome Sequence: a Novel Gene Arrangement and its Phylogenetic Position within the Class Chromadorea.

Human gnathostomiasis is an emerging food-borne parasitic disease caused by nematodes in the genus Gnathostoma. In spite of their significance as pathogens, these parasites remain poorly understood at the molecular level. In the present study, we sequenced the mitochondrial (mt) genome of G. spinigerum, which infects a range of definitive hosts including dogs, cats, tigers, leopards and humans. The mt genome of G. spinigerum is 14,079 bp in size and shows substantial changes in gene order compared to other nematodes studied to date. Phylogenetic analyses of mt genome sequences by Bayesian inference (BI) revealed that the infraorder Gnathostomatomorpha (represented by G. spinigerum) is closely related to the infraorder Ascaridomorpha. G. spinigerum is the first species from the infraorder Gnathostomatomorpha for which a complete mt genome has been sequenced. The new data will help understand the evolution, population genetics and systematics of this medically important group of parasites.

Gnathostoma spinigerum infection in the upper lip of a Korean woman: an autochthonous case in Korea.

Autochthonous human gnathostomiasis had never been reported in the Republic of Korea. We report here a case of Gnathostoma spinigerum infection in a 32-year-old Korean woman, presumed to have been infected via an indigenous route. The patient had experienced a painful migratory swelling near the left nasolabial fold area of the face for a year, with movement of the swelling to the mucosal area of the upper lip 2 weeks before surgical removal of the lesion. Histopathological examinations of the extracted tissue revealed inflammation with heavy eosinophilic infiltrations and sections of a nematode suggestive of a Gnathostoma sp. larva. The larva characteristically revealed about 25 intestinal cells with multiple (3-6) nuclei in each intestinal cell consistent with the 3rd-stage larva of G. spinigerum. The patient did not have any special history of travel abroad except a recent trip, 4 months before surgery, to China where she ate only cooked food. The patient is the first recorded autochthonous case of G. spinigerum infection in Korea.

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.

[Detection and identification for Gnathostoma sp. in imported Monopterus albus].

OBJECTIVE: To inspect the third stage larvae of Gnathostoma in imported Monopterus albus, and identify its species. METHODS: Ten batches of M. albus imported to Shanghai were detected for nematode Gnathostoma from January 2010 to March 2011. Fifty-two M. albus imported from the Philippines (25), Indonesia (24) and Bangladesh (3) were sampled (3-10/batch), which were dissected, minced, and digested. The suspension was filtered with 10 mesh screen to take the deposit. The complete parasites were picked out under stereoscope followed by morphological identification. The rate and intensity of infection were calculated. Genomic DNA of Gnathostoma was extracted to amplify internal transcribed spacer region 2 (ITS-2) and cytochrome C oxidase subunit 1 (cox1) by PCR, the product of which was analyzed by electrophoresis and sequencing. The sequences were aligned with corresponding sequences in GenBank. RESULTS: The third stage larvae of Gnathostoma were detected in M. albus from Indonesia and Philippines with infection rate of 36.0% (9/25) and 50.0% (12/24) and average infectiosity of 7.8 (70/9) and 2.8 (34/12), respectively. No Gnathostoma was found in M. albus imported from Bangladesh. Under microscope, the larvae showed one cephalic bulb with 4 rings of hooklets on it, cross striations and small spines on the body surface. The front body spines were bigger and denser, while the rear spines were smaller and sparser. It had 1 cervical papilla and 4 cervical capsules. Morphological characteristics were similar to the third stage larvae of G. spinigerum. PCR results showed that the length of the ITS-2 and cox1 PCR products was 647 bp and 441 bp, respectively. Sequence alignment analysis showed that the two PCR products had 99%-100% consistency with G. spinigerum ITS-2 (GenBank Accession No. AB181155 and Z97175) and cox1 (GenBank Accession No. AY501388, AB180099, and AB551552). CONCLUSION: All the larvae detected in M. albus imported from the Philippines and Indonesia have been identified as G. spinigerum.

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.

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.

Environmental niche equivalency versus conservatism: quantitative approaches to niche evolution.

Environmental niche models, which are generated by combining species occurrence data with environmental GIS data layers, are increasingly used to answer fundamental questions about niche evolution, speciation, and the accumulation of ecological diversity within clades. The question of whether environmental niches are conserved over evolutionary time scales has attracted considerable attention, but often produced conflicting conclusions. This conflict, however, may result from differences in how niche similarity is measured and the specific null hypothesis being tested. We develop new methods for quantifying niche overlap that rely on a traditional ecological measure and a metric from mathematical statistics. We reexamine a classic study of niche conservatism between sister species in several groups of Mexican animals, and, for the first time, address alternative definitions of "niche conservatism" within a single framework using consistent methods. As expected, we find that environmental niches of sister species are more similar than expected under three distinct null hypotheses, but that they are rarely identical. We demonstrate how our measures can be used in phylogenetic comparative analyses by reexamining niche divergence in an adaptive radiation of Cuban anoles. Our results show that environmental niche overlap is closely tied to geographic overlap, but not to phylogenetic distances, suggesting that niche conservatism has not constrained local communities in this group to consist of closely related species. We suggest various randomization tests that may prove useful in other areas of ecology and evolutionary biology.

Identification of estuarine fish Dormitator latifrons as an intermediate host and Eleotris picta as a paratenic host for Gnathostoma binucleatum in Sinaloa, Mexico.

Gnathostomosis is a typical fish-borne zoonotic parasitosis and is currently a serious public health issue in Mexico. Among several Gnathostoma species present in wild animals in Mexico, Gnathostoma binucleatum is the only proven species responsible for human diseases, and the advanced third stage larvae (AL3) of G. binucleatum have been found in over 20 species of fish in this country. In Sinaloa State, two fish species, Dormitator latifrons and Eleotris picta, were heavily contaminated with G. binucleatum AL3. When we analyzed the relationship between the size of the fish and the density of infection with G. binucleatum AL3, the distribution patterns of AL3 were markedly different between these two fish species. Apparent size-dependent accumulation was observed in E. picta but not in D. latifrons, suggesting that E. picta is a paratenic host whereas D. latifrons is a second intermediate host.

Relationships of Afroablepharus Greer, 1974 skinks from the Gulf of Guinea islands based on mitochondrial and nuclear DNA: patterns of colonization and comments on taxonomy.

Partial sequences of three mitochondrial DNA genes, 12S rDNA, 16S rDNA and cytochrome b, and one nuclear gene, c-mos, were used to assess the phylogenetic relationships of species belonging to the genus Afroablepharus from the volcanic islands of the Gulf of Guinea (West Africa) and neighboring continental Africa. Additionally, partial sequences of cytochrome b were used to compare levels of sequence divergence within populations. The three forms from São Tomé, Príncipe and Annobon (one per island) are genetically distinct, with high levels of divergence, supporting the recognition of a distinct species in each island. Populations within each island contain very low levels of genetic diversity. These three forms form a monophyletic group suggesting a single initial colonization followed by radiation to the other islands, possibly from São Tomé to Príncipe and Annobon. This is contrary to what was found in other reptiles from these islands such as Mabuya (sensu lato) and Hemidactylus, which colonized the islands multiple times. Assuming a molecular clock for cytochrome b of about 2% divergence per million years (usually applied to Sauria), the lineage on Annobon island exceeds the age of the island, thus casting further doubt on this widely used divergence estimate. Partial sequences of c-mos showed no variation within islands. Five to seven sites were variable among islands, which is a high value further supporting the treatment of each island form as a distinct species.

[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.

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.

A new species of Gnathostoma (Nematoda: Gnathostomatidae) in Procyon lotor hernandezii from Mexico.

Gnathostoma lamothei n. sp., inhabiting the stomach of Procyon lotor hernandezii Wagler, 1831, in Tlacotalpan, Veracruz State, and Rio Sapo, Oaxaca, Mexico, is described. This new species differs from all other congeners by having the posterior half of the body surface covered by rows of tiny round bosses instead of spines, or lacking ornamentations. Sequences of the ITS2 of the ribosomal DNA of G. lamothei n. sp. are compared with sequences of other species of the genus recorded in Mexico; they show a wide divergence (<50%) with Gnathostoma binucleatum Almeyda-Artigas, 1991, and Gnathostoma turgidum Stossich, 1902, and high similarity with Gnathostoma sp. I sequence (99.2%). On the basis of morphometric traits and sequences, previous records of Gnathostoma sp. I (=Gnathostoma procyonis of Almeyda-Artigas et al., 1994, not Chandler, 1942, and Gnathostoma neoprocyonis nomen nudem) in Mexico are referred to as the new species.