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.

Proteomic analysis identification of antigenic proteins in Gnathostoma spinigerum larvae.

Gnathostoma spinigerum is the causative agent of human gnathostomiasis. The advanced third stage larva (AL3) of this nematode can migrate into the subcutaneous tissues, including vital organs, often producing severe pathological effects. This study performed immuno-proteomic analysis of antigenic spots, derived from G. spinigerum advanced third stage larva (GSAL3) and recognized by human gnathostomiasis sera, using two-dimensional (2-DE) gel electrophoresis based-liquid chromatography/tandem mass spectrometry (LC/MS-MS), and followed by the aid of a database search. The crude GSAL3 extract was fractionated using IPG strips (pH 3-11NL) and followed by SDS-PAGE in the second dimension. Each gel was stained with colloidal Coomassie blue or was electro-transferred onto a nitrocellulose membrane and probed with gnathostomiasis human sera by immunoblotting. Individual Coomassie-stained protein spots corresponding to the antigenic spots recognized by immunoblotting were excised and processed using LC/MS-MS. Of the 93 antigenic spots excised, 87 were identified by LC/MS-MS. Twenty-seven protein types were found, the most abundant being Ascaris suum37. Six spots showed good quality spectra, but could not be identified. This appears to be the first attempt to characterize antigenic proteins from GSAL3 using a proteomic approach. Immuno-proteomics shows promise to assist the search for candidate proteins for diagnosis and vaccine/drug design and may provide better understand of the host-parasite relationship in human gnathostomiasis.

Increasing Risk Factors for Imported and Domestic Gnathostomiasis in the United States.

Gnathostomiasis is a foodborne zoonotic helminthic infection, commonly described in Asia and Latin America, which may follow the consumption of raw fish, eels, amphibians, and reptiles infected with muscle-encysted larvae of Gnathostoma species nematodes. After an inoculum of as little as one infective larva and an incubation period of months to years, most infections are characterized by intermittent migratory swellings due to subdermal larval migration. Less commonly, larval migration to the central nervous system may result in radiculomyelopathy or eosinophilic meningoencephalitis with high fatality rates; or larval migration to the eye with resulting blindness in untreated cases. Since the US now supports a zoonosis of Gnathostoma species with infective larvae encysted in imported and domestic fish and eels that may be consumed raw as exotic ethnic dishes, the objectives of this review were to describe the biology and life cycle of Gnathostoma nematodes and the behavioral risk factors for gnathostomiasis; and to describe the clinical manifestations, diagnosis, management, and prevention of human gnathostomiasis. Since the eradication of gnathostomiasis is very unlikely given the global distribution of Gnathostoma nematodes and the increasingly exotic culinary tastes of US residents and travelers to endemic regions, the only effective strategies for gnathostomiasis include: (1) educating citizens in the US and travelers abroad in endemic areas that fish, eels, frogs, snakes, and chicken must be cooked thoroughly first and not eaten raw or marinated; and (2) seeking medical care immediately for evaluation of migratory subcutaneous swellings. The combination of international travel and increased immigration from Asia and Latin America to the US has resulted in greater popularity of exotic ethnic cuisine, especially raw seafood dishes. The ethnic cuisine industry is supported by domestic aquaculture that produces fish-farmed tilapia and trout, and by increased importation of live freshwater species, such as Asian swamp eels (Monopterus spp.).1 Although raw seafood dishes are typically prepared with saltwater species, freshwater species, which harbor more parasites, are also used in these dishes, such as limejuice marinated tilapia or trout ceviche and eel-sashimi and sushi.2 In 2014, biologists from the US Geological Survey detected Gnathostoma species infective-stage larvae in nearly 30 percent of imported Monopterus species Asian swamp eels and in 4.5 percent of locally-caught Monopterus species freshwater swamp eels in three states.1 The investigators concluded that consumption of imported swamp eels from Gnathostoma-endemic regions of Asia could transmit gnathostomiasis to humans in the US.1 In addition, the release of live imported swamp eels or the disposal of their offal after filleting has introduced more Gnathostoma larvae into open and fish-farmed freshwaters infecting more native species.1 Since the US has all of the components to support Gnathostoma's life cycle, a zoonosis of infective Gnathostoma species, such as G. spinigerum and others, has become established in the US and will increase the risks of gnathostomiasis in humans consuming native, wildcaught, or farmed fish in ethnic dishes.1 As a result, the objectives of this review were to describe the biology and life cycle of Gnathostoma nematodes and the behavioral risk factors for gnathostomiasis; and to describe the clinical manifestations, diagnosis, management, and prevention of human gnathostomiasis.

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.

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.

Description of advanced third-stage larvae of Gnathostoma lamothei Bertoni-Ruiz et al. 2005 (Nematoda: Gnathostomatidae) from experimental hosts and contributions to its life cycle.

The advanced third-stage larvae (AdvL(3)) of Gnathostoma lamothei was obtained from experimental hosts. Frogs Lithobates heckscheri and snakes Nerodia fasciata pictiventris were compatible hosts allowing optimal larval development. AdvL(3) are 4,487.94 µm long, have two lateral cervical papillae between rows 10 and 16 and an excretory pore at row 23. The average counts of the cephalic bulb hooklets from the four rows are 39.3, 43.3, 44.2, and 47.3. Larvae show an esophagus that represents 40 % of the body width. These findings indicate that amphibians and reptiles could be involved as G. lamothei natural hosts; nevertheless, their role as etiological agents of human gnathostomiasis is uncertain. This paper reports for the first time the taxonomic description of G. lamothei AdvL(3) obtained from experimental hosts and contributes to the understanding of its life cycle.

Neurognathostomiasis, a neglected parasitosis of the central nervous system.

Gnathostomiasis is a foodborne zoonotic helminthic infection caused by the third-stage larvae of Gnathostoma spp. nematodes. The most severe manifestation involves infection of the central nervous system, neurognathostomiasis. Although gnathostomiasis is endemic to Asia and Latin America, almost all neurognathostomiasis cases are reported from Thailand. Despite high rates of illness and death, neurognathostomiasis has received less attention than the more common cutaneous form of gnathostomiasis, possibly because of the apparent geographic confinement of the neurologic infection to 1 country. Recently, however, the disease has been reported in returned travelers in Europe. We reviewed the English-language literature on neurognathostomiasis and analyzed epidemiology and geographic distribution, mode of central nervous system invasion, pathophysiology, clinical features, neuroimaging data, and treatment options. On the basis of epidemiologic data, clinical signs, neuroimaging, and laboratory findings, we propose diagnostic criteria for neurognathostomiasis.

Gnathostoma binucleatum: pathological and parasitological aspects in experimentally infected dogs.

Lesions and antibody kinetics produced by inoculation of Gnathostoma binucleatum larvae into dogs are described, as well as the morphology of the recovered parasites. In four out of five infected bitches parasite phases were found in the stomach. Only one bitch eliminated eggs and adult parasite phases in feces. In this bitch, the prepatency period lasted 22 weeks and the patency period 14 weeks. Necropsy results showed a copiously vascularized 8-cm diameter fibrous nodule lodged in the greater curvature of the stomach. Two bitches that eliminated no eggs showed 1- to 2-cm diameter nodules on the gastric wall, with five juvenile phases in each. One bitch that eliminated no eggs and exhibited no gastric nodules showed juvenile parasites on the gastric wall. Results confirm dogs as definitive hosts of this parasite. New data on the pathological and parasitological aspects of canine gnathostomosis are presented.

Gnathostoma hispidum infection in a Korean man returning from China.

Human Gnathostoma hispidum infection is extremely rare in the world literature and has never been reported in the Republic of Korea. A 74-year-old Korean man who returned from China complained of an erythematous papule on his back and admitted to our hospital. Surgical extraction of the lesion and histopathological examination revealed sections of a nematode larva in the deep dermis. The sectioned larva had 1 nucleus in each intestinal cell and was identified as G. hispidum. The patient recalled having eaten freshwater fish when he lived in China. We designated our patient as an imported G. hispidum case from China.

Elevated Na+/K+-ATPase responses and its potential role in triggering ion reabsorption in kidneys for homeostasis of marine euryhaline milkfish (Chanos chanos) when acclimated to hypotonic fresh water.

The milkfish (Chanos chanos) is an economic species in Southeast Asia. In Taiwan, the milkfish are commercially cultured in environments of various salinities. Na(+)/K(+)-ATPase (NKA) is a key enzyme for fish iono- and osmoregulation. When compared with gills, NKA and its potential role were less examined by different approaches in the other osmoregulatory organs (e.g., kidney) of euryhaline teleosts. The objective of this study was to investigate the correlation between osmoregulatory plasticity and renal NKA in this euryhaline species. Muscle water contents (MWC), plasma, and urine osmolality, kidney histology, as well as distribution, expression (mRNA and protein), and specific activity of renal NKA were examined in juvenile milkfish acclimated to fresh water (FW), seawater (SW 35 per thousand), and hypersaline water (HSW 60 per thousand) for at least two weeks before experiments. MWC showed no significant difference among all groups. Plasma osmolality was maintained within the range of physiological homeostasis in milkfish acclimated to different salinities, while, urine osmolality of FW-acclimated fish was evidently lower than SW- and HSW-acclimated individuals. The renal tubules were identified by staining with periodic acid Schiff's reagent and hematoxylin. Moreover, immunohistochemical staining showed that NKA was distributed in the epithelial cells of proximal tubules, distal tubules, and collecting tubules, but not in glomeruli, of milkfish exposed to different ambient salinities. The highest abundance of relative NKA alpha subunit mRNA was found in FW-acclimated milkfish rather than SW- and HSW-acclimated individuals. Furthermore, relative protein amounts of renal NKA alpha and beta subunits as well as NKA-specific activity were also found to be higher in the FW group than SW and the HSW groups. This study integrated diverse levels (i.e., histological distribution, gene, protein, and specific activity) of renal NKA expression and illustrated the potential role of NKA in triggering ion reabsorption in kidneys of the marine euryhaline milkfish when acclimated to a hypotonic FW environment.

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.

Prevalence of Gnathostoma spinigerum infection in wild and cultured swamp eels in Vietnam.

Human infections with Gnathostoma spinigerum frequently occur in southern Vietnam. Previous investigations have implicated infected swamp eels (Monopterus albus) as an important source of infection to humans. Because aquaculture of M. alba is an important farming activity in Vietnam, a 2-yr study was carried out to assess the relative importance of farmed and wild eels as potential sources of gnathostome infections in humans. Eels sold for public consumption in markets in southern provinces were examined between November 2005 and August 2007. Although no infections were detected in 1,020 cultured eels and 1,021 wild-caught eels collected from November 2005 to September 2006, larval G. spinigerum (AL3) infections were first detected in September 2006 in 28 of 230 wild-caught eels (12.2%) obtained from markets in Long An province and the Hoc Mon district of Ho Chi Minh City. Subsequently, monthly surveillance of wild-caught eels from these markets was carried out through August 2007. Prevalence of AL3 varied monthly, ranging from 0.8 to 19.6%. Both prevalence and infection intensity were higher during the latter part of the rainy season (August- October). These results demonstrate that potentially zoonotic G. spinigerum larvae are common in wild eels in southern Vietnam and present a risk to consumers of raw fish dishes, especially during the annual rainy season. This information could help target public health education efforts in the region. The basis for the seasonal variation on eel infections is not known, but may be related to climate effects (flooding, higher temperatures) on intermediate host species ecology.

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.

All vertebrates started out with a glial blood-brain barrier 4-500 million years ago.

All extant vertebrates have a blood-brain barrier (BBB), a specialized layer of cells that controls molecular traffic between blood and brain, and contributes to the regulation (homeostasis) of the brain microenvironment. Such homeostasis is critical for the stable function of synapses and neural networks. The barrier is formed by vascular endothelial cells in most groups, but by perivascular glial cells (astrocytes) in elasmobranch fish (sharks, skates, and rays). It has been unclear which is the ancestral form, but this information is important, as it could offer insights into the roles of the endothelium and perivascular glia in the modern mammalian BBB. We have used electron microscopic techniques to examine three further ancient fish groups, with intravascular horseradish peroxidase as permeability tracer. We find that in bichir and lungfish the barrier is formed by brain endothelial cells, while in sturgeon it is formed by a complex perivascular glial sheath, but with no detectable tight junctions. From their BBB pattern, and position on the vertebrate family tree, we conclude that the ancestral vertebrate had a glial BBB. This means that an endothelial barrier would have arisen independently several times during evolution, and implies that an endothelial barrier gave strong selective advantage. The selective advantage may derive partly from greater separation of function between endothelium and astrocytic glia. There are important implications for the development, physiology, and pathology of the mammalian BBB, and for the roles of endothelium and glia in CNS barrier layers.

Estuarine fish and turtles as intermediate and paratenic hosts of Gnathostoma binucleatum in Nayarit, Mexico.

Human gnathostomosis is a severe public health problem in the State of Nayarit, Mexico. Between 1995 and 2005, the registration of human cases numbered 6,328, which makes it one of the largest focal points of the disease in the country. The present study determined the presence of natural hosts of Gnathostoma binucleatum larvae at the Laguna de Agua Brava in Nayarit, Mexico. A total of 5,450 fish and 247 turtles were sampled. Muscular tissue was ground and observed against the light using a 100-W lamp to identify advanced third-stage larvae. The estuarine species Cathorops fuerthii, Pomadasys macracanthus, Mugil curema, and Dormitator latifrons were found positive for presence of larvae, and annual prevalence was 4.8, 1.83, 2.16, and 4.0%, respectively. The species Oreochromys aureus and Chanos chanos were negative. The species of estuarine turtles Kinosternum integrum and Trachemys scripta were positive with annual prevalence of 79.1 and 52.5%, respectively. The criteria of identification of the Gnathostoma species were: mean number of nuclei in intestinal larval cells (2.3), larval morphometry with optic microscopy, larval morphometry with scanning electron microscopy, and number and sequence of ribosomal deoxyribonucleic acid of adult parasites obtained from experimental infection in dogs. The estuarine fish Pomadasys macracanthus and Mugil curema are reported as intermediate hosts for the first time and likewise the estuarine turtle Kinosternon integrum as a paratenic host.

Sensitivity of mottled sculpins (Cottus bairdi) and rainbow trout (Onchorhynchus mykiss) to acute and chronic toxicity of cadmium, copper, and zinc.

Studies of fish communities of streams draining mining areas suggest that sculpins (Cottus spp.) may be more sensitive than salmonids to adverse effects of metals. We compared the toxicity of zinc, copper, and cadmium to mottled sculpin (C. bairdi) and rainbow trout (Onchorhynchus mykiss) in laboratory toxicity tests. Acute (96-h) and early life-stage chronic (21- or 28-d) toxicity tests were conducted with rainbow trout and with mottled sculpins from populations in Minnesota and Missouri, USA, in diluted well water (hardness = 100 mg/L as CaCO3). Acute and chronic toxicity of metals to newly hatched and swim-up stages of mottled sculpins differed between the two source populations. Differences between populations were greatest for copper, with chronic toxicity values (ChV = geometric mean of lowest-observed-effect concentration and no-observed-effect concentration) of 4.4 microg/L for Missouri sculpins and 37 microg/L for Minnesota sculpins. Cadmium toxicity followed a similar trend, but differences between sculpin populations were less marked, with ChVs of 1.1 microg/L (Missouri) and 1.9 microg/L (Minnesota). Conversely, zinc was more toxic to Minnesota sculpins (ChV = 75 microg/L) than Missouri sculpins (chronic ChV = 219 microg/L). Species-average acute and chronic toxicity values for mottled sculpins were similar to or lower than those for rainbow trout and indicated that mottled sculpins were among the most sensitive aquatic species to toxicity of all three metals. Our results indicate that current acute and chronic water quality criteria for cadmium, copper, and zinc adequately protect rainbow trout but may not adequately protect some populations of mottled sculpins. Proposed water quality criteria for copper based on the biotic ligand model would be protective of both sculpin populations tested.

[Parasitic dead-end: update]. / Impasses parasitaires: quoi de neuf?

Parasitic dead-ends occur when a parasite is unable to establish a permanent interaction in an unnatural host. Although the likelihood of successful reproduction by the pathogenic agent is nul, parasitic dead-end heralds capture of new parasites and therefore expansion of the host range. Angiostrongyliasis due to A. cantonensis or A. costaricensis, anisakiasis, Ancylostoma caninum infection, gnathostomiasis and sparganosis are undoubtedly emerging zoonoses of particular medical interest. Prevention of these diseases relies on abstinence from eating raw meat from invertebrates or cold-blooded (poikilotherm) vertebrates (e.g. used in exotic dishes). These guidelines must be included in recommendations to travelers.

Outward migration of Gnathostoma spinigerum in interferon alpha treated hepatitis C patient.

After the first dose injection of pegylated interferon alpha-2b (Peg-IFN alpha-2b) to a HCV infected Thai woman, she developed cyclic painful swelling nodules on right upper quadrant of abdomen and right anterior lower chest wall. The nodules subsided spontaneously within 1-2 days but were recurrent after every Peg-IFN alpha-2b injection. She also experienced acute urticaria. After nine months of therapy, an immature male of G. spinigerum migrated out from the skin nodule shortly after a Peg-IFN alpha-2b injection as scheduled. The worm showed a head-bulb bearing 8 transverse rows of spines which indicated immature stage. It had well defined four pairs of caudal papillae on posterior body part which were used to identify male gender. Painful migratory swelling and urticaria disappeared after the parasite was removed. She was continually treated and had sustained both virological and biochemical responses to HCV treatment. This case demonstrates that the outward migration of G. spinigerum may be stimulated by the injection of Peg-IFN alpha-2b.

Fish as the natural second intermediate host of Gnathostoma spinigerum.

Gnathostomiasis is a helminthic disease most frequently occurring in Thailand. Human infections are usually found to be caused by Gnathostoma spinigerum, although five species of the genus Gnathostoma exist in Thailand, and three of these are capable of infecting man. In Thailand, 47 species of vertebrates--fish (19), frogs (2), reptiles (11), birds (11) and mammals (4)--have been reported to serve naturally as the second intermediate (and/or paratenic) hosts of G. spinigerum. Of these, fish, especially swamp eels (Monopterus albus), were found to be the best second intermediate/paratenic hosts: they had the highest prevalence rate and the heaviest infection intensity. However, the scientific names of these fish have been revised from time to time. Therefore, for clarity and consistency, we have summarized the current scientific names of these 19 species of fish, together with their illustrations. We describe one additional fish species, Systomus orphoides (Puntius orphoides), which is first recorded as a naturally infected second intermediate host of G. spinigerum.