Morphological and molecular characterisation of Heliconema hainanensis sp. nov. (Spirurina: Physalopteridae) from congers in the South China Sea, with a key to the species of Heliconema

Heliconema hainanensis sp. nov. collected from Uroconger lepturus (Richardson) (Anguilliformes: Congridae), Muraenesox cinereus (Forsskål) and Congresox talabonoides (Bleeker) (Anguilliformes: Muraenesocidae) in the South China Sea was described using light and scanning electron microscopy. The new species differs from its congeners by the following morphology: pseudolabia, the number and arrangement of caudal papillae (4 pairs of pedunculate precloacal papillae arranged in 2 groups of 2 and 2 pairs and 6 pairs of pedunculate postcloacal papillae arranged in 4 groups of 1, 2, 1 and 2 pairs), the length of spicules [left spicule 0.51-0.69 mm, right spicule 0.20-0.27 mm, spicule (right:left) ratio 1:2.20-2.69] and the morphology of the female tail tip. In addition, specimens of the new species collected from the three different hosts and specimens of an unidentified species of Heliconema collected from U. lepturus were characterised using molecular methods by sequencing the internal transcribed spacer (ITS) of ribosomal DNA. Analyses and comparison of the ITS sequence of H. hainanensis sp. nov. with Heliconema sp. support the validity of the new species based on morphological observations. An identification key to the species of Heliconema is also provided.

Feasibility of using papaya skin extract for digestion of swamp eel viscera for harvesting infective stage larva of Gnathostoma spp.

In this study, we reported the feasibility of using papaya skin extract (Carica papaya L.) as an alternative to enzyme pepsin in harvesting Gnathostoma spinigerum third-stage larvae. From experimental digestion, we found that the different numbers of recovered larvae between papaya skin extract and pepsin were not statistically significant (p >0.05). When the derived larvae from pepsin and papaya skin extract digestion were cultivated in BME medium for 7 days, the survival rates were not significantly different either (p >0.05). Thus, papaya skin extract might be another choice for recovering Gnathostoma spinigerum third-stage larvae.

Gnathostoma infection in fish caught for local consumption in Nakhon Nayok Province, Thailand I. Prevalence and fish species.

Between August 2000 and August 2001, 12,216 fish of 73 species were purchased from several local markets in Nakhon Nayok Province, Thailand, and examined for the presence of Gnathostoma larvae. Almost all species were fresh-water fish that had grown naturally, rather than raised commercially. Eight species were found to be infected with gnathostome larvae. The overall prevalence was 5.1% (626/12,216) and a total of 5,969 larvae was recovered. The highest rate of infection (30.1 %) was found in Monopterus albus (swamp eel). The rates in the remaining infected fish were as follows: Anabas testudineus (climbing perch) 7.7%, Channa striata (striped snake-head fish) 7.4%, Clarius macrocephalus (Gunther's walking catfish) 6.7%, Channa micropeltes (giant snake-head fish) 5.1%, Channa lucius (blotched snake-head fish) 4.0%, Clarius batrachus (Batrachian walking catfish) 1.4%, and Ompok krattensis (butter sheatfish) 0.6%. The mean number of larvae/fish was highest in swamp eels (10.0 larvae/eel), and the maximum number of 698 larvae was recovered from one eel. The body sizes of the recovered G. spinigerum advanced third-stage larvae were 2.70-5.10 mm in length (average, 3.97+/-0.50 mm) and 0.29-0.60 mm in width (average, 0.40+/-0.04 mm). The average number of cephalic hooklets of the larvae from rows 1 to 4 were 41.8+/-0.5 (range, 40-43), 43.6+/-0.6 (range, 42-45), 46.1+/-0.9 (range, 44-48) and 49.3+/-0.7 (range, 48-51), respectively.

Pineapple juice for digestion of swamp eel viscera for harvesting infective-stage larva of Gnathostoma spp.

Third-stage larvae were used as antigen in the diagnosis of gnathostomiasis in Western blot analysis. Normally, the larvae were obtained from digestion of eel's liver (Fluta alba) by the enzyme pepsin. We used pineapple juice (Ananus comosus) instead of enzyme pepsin in harvesting Gnathostoma spinigerum third-stage larvae. The difference in recovered larvae numbers, between pineapple juice and pepsin, were not statistically significantly different (p>0.05). The larvae from pepsin and pineapple juice digestion were cultivated on BME for 7 days; the survival rates were not significantly different (p>0.05). Thus, pineapple juice is another enzyme of choice for recovering Gnathostoma spinigerum third-stage larvae.

Prevalence and intensity of third stage Gnathostoma spinigerum larvae in swamp eels sold in three large markets in Bangkok, Thailand.

Gnathostoma spinigerum is a common human tissue parasite in Thailand. The swamp eel is the major intermediate or paratenic host for this parasite. The high prevalence of the infective third stage larvae (L3) of this parasite in the rainy season has been noted in previous studies. During June 1999 (rainy season), we performed a cross-sectional survey of the prevalence and the intensity of G. spinigerum L3s in the livers of swamp eels that were obtained from three large Bangkok markets (Klong Toey, Pran Nok, and Tevej). Of a total of 785 livers, G. spinigerum L3s were found in 97: an infection rate of 12%. The prevalence rates in Klong Toey, Pran Nok, and Tevej markets were 13%, 10% and 14% respectively. There was no significant difference in the proportion of infected eels between the markets (p > 0.05). The intensity of L3 burden in the livers ranged from 1-17 larvae, with an average of 2.60 +/- 0.24 larvae (mean +/- SE). In this study a high prevalence of G. spinigerum L3s was found; there was no significant difference in the intensity of larvae in swamp eels between markets. These findings emphasize the importance of public health education: people need to be aware of the dangers of consuming raw or undercooked food.

Lack of efficacy of quinine and artemether against advanced third-stage larvae of Gnathostoma spinigerum in vitro.

The efficacy of quinine and artemether--the effective blood schizontocide in malarial treatment--has been in vitro tested with the advanced third-stage larvae of Gnathostoma spinigerum. All larvae were collected from freshwater eel (Fluta alba) and exposed to the culture medium, each containing either quinine dihydrochloride or artemether at a final concentration of 20 microg/ml and 0.5 microg/ml, respectively for 21 consecutive days. Larval motility was assessed daily and the topographical changes were assessed using scanning electron microscope after 21-days of drug exposure. All worms moved actively for 21 days of study period and no change in surface ultrastructure was observed. Quinine and artemether at these concentrations have no effect on movement and topographical changes on the advanced third-stage larvae of this parasite.

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.

Gnathostome infection in swamp eels, Fluta alba, in central Thailand.

To investigate the distribution of gnathostome worms in central Thailand, the infective larvae of Gnathostoma spp were examined from the flesh and liver of swamp eels, Fluta alba. Seven hundred and eighty-eight eels were purchased from markets in 11 provinces; Ang Thong (30), Ayutthaya (36), Chachoengsao (30), Lop Buri (30), Nakhon Nayok (437), Pathum Thani (30), Prachin Buri (48), Ratchaburi (53), Saraburi (30), Samut Prakan (30) and Suphan Buri (34). The highest rate of gnathostome infection was observed in swamp eels from Nakhon Nayok (68.7%). The infection rates in Ayutthaya, Ang Thong, Prachin Buri, Ratchaburi, Saraburi and Lop Buri were 33.3%, 26.7%, 25.0%, 18.9%, 13.3% and 10.0% respectively. Gnathostome larvae were not found in swamp eels from Chachoengsao, Pathum Thani, Samut Prakan and Suphan Buri. Among the 9,573 larvae recovered, almost all were the advanced third stage larvae of G. spinigerum, except one larva from Nakhon Nayok and two larvae from Ratchaburi which were identified as the advanced third stage larvae of G. vietnamicum and G. hispidum respectively. This study is the first report of swamp eels as natural intermediate hosts of G. vietnamicum and G. hispidum.

Seasonal variation in the intensity of Gnathostoma larvae in swamp eels (Fluta alba) sold in a local market in Bangkok.

The viscera of swamp eels were obtained from a local market in Bangkok twice a month from June 1996 to May 1997. The livers were separated, weighed and counted. Gnathostome larvae were recovered from the livers by the digestion technic, examined, identified, and counted. A total of 12,278 Gnathostoma larvae were obtained from 18,561.1 g (15,264 pieces) of eel livers. The overall average number of larvae/g liver and the overall average number of larvae/liver are 0.91 and 0.94, respectively. The greatest number of larvae/g liver (on average) was in December (high levels of infection during the months of October to December) whereas the lowest was in April (lowest levels of infection during the months of March to April). Thus there was a marked decrease in the average number of larvae/g liver during January to April, which then started to rise in May. This finding suggests that the level of infection abruptly decreases soon after the completion of the rainy season, starts to rise when the rain has come, and reaches its peak when the amount of rainfall is highest. More than 99% of the total gnathostome larvae recovered were identified to be G. spinigerum, and 25.4% of the entire larvae recovered bore variant or abnormal cephalic hooklets. The most common unusual feature was that there were extra rudimentary hooklets above row one, below row four and in between the four rows of hooklets which comprised 21.4%. In addition, the body size and the number of cephalic hooklets of G. spinigerum are also discussed.

A new type of advanced third-stage larvae of the genus Gnathostoma in freshwater eels, Fluta alba, from Nakhon Nayok, central Thailand.

Five advanced third-stage larvae of a newly identified type of genus Gnathostoma were collected from freshwater eels, Fluta alba, which were purchased at a market in Nakhon Nayok, central Thailand. The most remarkable characteristic of the newly identified larvae was the larger body size compared with any other larva of Gnathostoma spp. They were also distinguishable from other species by the shape of their hooklets, which branched in a complex manner at the base: this had not been previously observed in any other larval Gnathostoma. The newly described larvae had an average number of 44.5, 45.0, 49.0 and 55.1 hooklets on the head-bulb from the first to the fourth rows, respectively, which were comparable to those of larval G. spinigerum. However, the average number of nuclei in each intestinal cell was 2.21 and fewer than those of the larvae of G. spinigerum. These results suggest that the new type of larvae belong to either G. vietnamicum, G. malaysiae, or constitute a new species of the genus Gnathostoma.

Gnathostomiasis in Thailand: a survey on intermediate hosts of Gnathostoma spp. with special reference to a new type of larvae found in Fluta alba.

To clarify current status of gnathostomiasis in Thailand, a survey on intermediate hosts has been carried out at various localities since 1987. It was found that Fluta alba (Fresh water eel) as well as Channa striata (snake-headed fish) might be important in playing a role of transmitting the infection either among humans or reservoir animals. During the three years from 1987 to 1989, larvae of Gnathostoma spinigerum were found in 80-100% of F. alba obtained from markets in Nakhon Nayok, with a maximum recovery of 2,582 larvae per eel. Among larvae found in these eels, five were peculiar in possessing four rows of hooklets with complicated branches at the base. Epithelial cells of the intestine of these larvae contained 1-2 nuclei. These observations indicate that the larvae are different from those of reported species of Gnathostoma from Thailand including G. spinigerum, suggesting a possibility of the advanced third-stage larvae of G. malaysiae.