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.

Histopathological changes in the liver and stomach of Didelphis virginiana (Didelphimorphia: Didelphidae) during natural infection with Gnathostoma turgidum (Nematoda: Gnathostomidae).

Gnathostoma turgidum is a nematode parasite that exploits the stomach of Virginian opossums, Didelphis virginiana, in Latin America. The opossum is the definitive host of G. turgidum in the wild. Intrahepatic growth and maturation of the parasite, subsequent migration to the stomach and spontaneous expulsion are common. However, the histopathological lesions caused by G. turgidum are poorly described. A better understanding of the life cycle of this parasite and the pathological changes in natural host-parasite interactions could help to clarify the progression of human infections caused by Gnathostoma binucleatum. The aim of this work was to study morphological changes in the liver and stomach of D. virginiana during natural infection and adult worm expulsion. Three opossums naturally infected with G. turgidum were captured from an endemic area of gnathostomosis. Three uninfected opossums captured from a non-endemic area were used as controls. The opossums were sacrificed at different stages of infection (March, May and December), and a histopathological study of their livers and stomachs was conducted. Injuries in livers were observed by histopathology - areas of necrosis and collagen septa were identified. Parasites caused nodules with necrosis on the periphery of lesions, and collagen fibres were also observed in stomachs. Collagen septa may be caused by antigenic remains of the parasite. Further immunological studies are necessary to verify that stimulation is caused by these factors.

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.

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.

Positivity and intensity of Gnathostoma spinigerum infective larvae in farmed and wild-caught swamp eels in Thailand.

From July 2008 to June 2009, livers of the swamp eels (Monopterus alba) were investigated for advanced third-stage larvae (AL3) of Gnathostoma spinigerum. Results revealed that 10.2% (106/1,037) and 20.4% (78/383) of farmed eels from Aranyaprathet District, Sa Kaeo Province and those of wild-caught eels obtained from a market in Min Buri District of Bangkok, Thailand were infected, respectively. The prevalence was high during the rainy and winter seasons. The infection rate abruptly decreased in the beginning of summer. The highest infection rate (13.7%) was observed in September and absence of infection (0%) in March-April in the farmed eels. Whereas, in the wild-caught eels, the highest rate (30.7%) was observed in November, and the rate decreased to the lowest at 6.3% in March. The average no. (mean±SE) of AL3 per investigated liver in farmed eels (1.1±0.2) was significantly lower (P=0.040) than those in the caught eels (0.2±0.03). In addition, the intensity of AL3 recovered from each infected liver varied from 1 to 18 (2.3±0.3) in the farmed eels and from 1 to 47 (6.3±1.2) in the caught eels, respectively. The AL3 intensity showed significant difference (P=0.011) between these 2 different sources of eels. This is the first observation that farmed eels showed positive findings of G. spinigerum infective larvae. This may affect the standard farming of the culture farm and also present a risk of consuming undercooked eels from the wild-caught and farmed eels.

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

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

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.

Complete mitochondrial genomes of Gnathostoma nipponicum and Gnathostoma sp., and their comparison with other Gnathostoma species.

Gnathostomiasis is a foodborne zoonotic parasitosis caused by Gnathostoma nematodes. It has caused significant public problems worldwide, but its molecular biology is limited. The purpose of this study was to decode the complete mitochondrial (mt) genomes of Gnathostoma nipponicum and Gnathostoma sp., and compare their mt sequences with other Gnathostoma species. The complete mt genome sequences were amplified by long-range PCR and determined by subsequent primer walking. The complete mt genomes of G. nipponicum and Gnathostoma sp. were 14,093bp and 14,391bp, respectively. Both of the two mt genomes contain 12 protein-coding genes (PCGs), 2 ribosomal RNA genes and 22 transfer RNA genes. The gene order and transcription direction are the same as G. spinigerum and G. doloresi. The sequence difference across the entire mt genomes varied from 14.4% to 18.2% between G. nipponicum, Gnathostoma sp., G. spinigerum and G. doloresi of Japan and China isolates. Phylogenetic analyses by Bayesian inference (BI) using concatenated amino acid sequences of 12 PCGs showed that G. nipponicum and Gnathostoma sp. are two distinctive species of Gnathostoma, and G. nipponicum are more closely related to Gnathostoma sp. than to G. spinigerum. The mtDNA datasets provide abundant resources of novel markers, which can be used for the studies of molecular epidemiology and diagnosis of Gnathostoma spp.

Spontaneous cure after natural infection with Gnathostoma turgidum (Nematoda) in Virginia opossums (Didelphis virginiana).

Seasonality of the nematode Gnathostoma turgidum in Virginia opossums (Didelphis virginiana) in the wild has been reported; however, the mechanisms involved in deworming are unknown. We monitored the parasitologic and biologic changes in four Virginia opossums naturally infected with G. turgidum by coproparasitologic examination and abdominal ultrasonography. Eggs became detectable in the feces of opossums in May, peaked in July and August, and suddenly decreased in October. Adults of G. turgidum were expelled in the feces mainly in September. Ultrasonography of the liver showed slight damage during May. Lesions in the stomach appeared in April and persisted until September. The abnormalities of the liver and stomach were resolved in November. These data suggest that G. turgidum is likely expelled as a result of host immunologic mechanisms, although termination of a natural life span cannot be definitively excluded.

Pathological and parasitological traits in experimentally infected cats with Gnathostoma binucleatum (Spirurida: Gnathostomatidae).

This study aims to describe some of the unknown pathological and parasitological traits of experimental feline gnathostomosis. Thirteen female cats were orally inoculated with 30 advanced third-stage Gnathostoma binucleatum larvae and were euthanized at various post-infection (p.i.) periods. Clinically, the cats presented with nausea, vomiting, abdominal pain and other nonspecific signs. None of the cats shed eggs in their fecal matter. One cat, euthanized at 6 months p.i., developed a fibrous vascular nodule 2-3 cm in diameter within its gastric wall. The nodule contained caverns filled with mucous and bloody fluid as well as a juvenile worm. The histological characteristics of the nodule were observed, and the morphology of the juvenile worm was revealed using scanning electron microscopy. Another cat, euthanized at 10 months p.i., was found to have a larva within its diaphragm. Infected cats developed increased antibody titers against antigens of G. binucleatum adults and larvae beginning in the first month p.i., and these titers were maintained until the end of the experiment, suggesting the presence of undetected migrating larvae. The low number of cats with parasites and poor development of the parasites found suggest that cats have a low susceptibility to infection by G. binucleatum and cast doubt on the importance of domestic cats in maintaining the biological cycle of this parasite in nature.