Infection Status of Gnathostoma spinigerum Larvae in Asian Swamp Eels,Monopterus albus,Purchased from Local Markets in Cambodia

Present study was performed to know the infection status of Gnathostoma sp. larvae in swamp eels from Cambodia. We purchased total 30 Asian swamp eels, Monopterus albus, from local markets in Pursat and Takeo Provinces and Phnom Penh on May and November 2017 and May 2018. All collected eels were transferred to our laboratory with ice and each of them was examined by artificial digestion method. A total of 15 larval gnathostomes (1–5 larvae) were detected from 55.6% (5/9) swamp eels in Pursat Province. No larval gnathostomes were found in 21 swamp eels in Takeo Province and Phnom Penh. The advanced third-stage larvae (AdL₃) detected were 2.575–3.825 (3.250) mm in length and 0.375–0.425 (0.386) mm in width. They had the characteristic head bulb (av. 0.104×0.218 mm) with 4 rows of hooklets, long muscular esophagus (1.048 mm), and 2 pairs of cervical sacs (0.615 mm). The number of hooklets in 4 rows on the head bulb was 41, 44, 47, and 50. In scanning electron microscopy, characteristic features were 4 rows of hooklets on the head bulb, cervical papillae, tegumental spines regularly arranged in transverse striations, and anus. The larval gnathostomes were identified as AdL₃ of Gnathostoma spinigerum based on the morphological characters. By the present study, it has been confirmed that G. spinigerum larvae are infected in Asian swamp eels, M. albus, in Pursat Province, Cambodia.     

Detection of Gnathostoma spinigerum Third-Stage Larvae in Snakeheads Purchased from a Central Part of Myanmar

To examine the infection status of freshwater fish with Gnathostoma spp. larvae in Myanmar, we purchased 15 snakeheads, Channa striatus, from a local market in a suburban area of Naypyidaw, the new capital city. Two larval gnathostomes were collected using an artificial digestion technique, and observed by a light microscope and a scanning electron microscope. The size of an intact larva was 2.65 mm long and 0.32 mm wide. The characteristic morphology of the larvae included the presence of a long esophagus (0.80 mm long), 2 pairs of cervical sacs (0.43 mm long), and a characteristic head bulb with 4 rows of hooklets. The number of hooklets in the 1st, 2nd, 3rd, and 4th row was 45, 48, 50, and 52, respectively. Based on these morphological characters, the larvae were identified as the advanced 3rd-stage larvae of Gnathostoma spinigerum. This is the first report of detection of G. spinigerum 3rd-stage larvae in the central part of Myanmar. Our study suggests that intake of raw meat of snakehead fish in Myanmar may result in human gnathostomiasis.     

Larval Gnathostoma hispidum detected in the red banded odd-tooth snake, Dinodon rufozonatum rufozonatum, from China

A total of 205 larval gnathostomes were collected from 18 (22.5%) of 80 red banded odd-tooth snakes, Dinodon rufozonatum rufozonatum, which had been smuggled from China and confiscated at Customs in Busan, Republic of Korea. In order to identify the species, some of the larvae were observed by a light microscope and a scanning electron microscope (SEM). The larvae were 2.18 x 0.29 mm in average size, and had a pair of lips at the anterior end, a muscular esophagus, 2 pairs of cervical sacs, and brownish intestines. The head bulb was characteristically equipped with 4 rows of hooklets; the average number of hooklets in each respective row was 38.6, 40.5, 41.5, and 43.7. In SEM views, the mouth evidenced a pair of lateral lips of equal size in a half-moon shape. Each lip featured a couple of labial papillae and a small amphid located between the 2 papillae. The hooklets on the head bulb had single-pointed, posteriorly-curved tips. The cuticular spines were larger and more densely distributed on the anterior part of the body, and decreased gradually in size and number toward the posterior body. On the basis of these morphological characteristics, the larvae were identified as the third stage larvae of Gnathostoma hispidum.     

Prevalence of Intestinal Protozoans among Schoolchildren in Suburban Areas near Yangon,Myanmar

Although intestinal protozoans are common etiologies of diarrhea, few studies have been conducted in Myanmar. This study planned to investigate the prevalence of Giardia lamblia, Entamoeba coli, Entamoeba histolytica, and Endolimax nana among schoolchildren and their guardians in suburban areas near Yangon, Myanmar. We performed a cross-sectional survey among schoolchildren and their guardians from 7 primary schools in South Dagon and Hlaing Thar Yar districts, Yangon, Myanmar. Stool samples were observed with a microscope after concentration technique and iodine staining. Total 821 stool samples, including 556 from schoolchildren and 265 from guardians, were examined. The median age was 6 years old for schoolchildren and 36 years old for guardians. A 53.1% of the school children and 14.6 % of the guardians were males. The overall prevalence of each intestinal protozoan species was as follows: 3.4% (28/821) for G. lamblia; 3.5% (29/821) for E. coli; 1.2% (10/821) for E. histoytica, and 3.0% for E. nana. This study showed that intestinal protozoans are common in primary schoolchildren and their guardians in suburban areas near Yangon, Myanmar. Health interventions, such as hand washing education, improvement of sanitation, and establishment of water purification systems are urgently needed in this area.     

High Prevalence of Enterobius vermicularis Infection among Schoolchildren in Three Townships around Yangon,Myanmar

In order to determine the status of Enterobius vermicularis infection among schoolchildren in suburban areas of Myanmar, 761 primary schoolchildren in 3 different townships around Yangon City were subjected to a survey using cello-tape anal swabs. The subjected schoolchildren were 383 boys and 378 girls who were 5-7 years of age. Only 1 anal swab was obtained from each child. The overall egg positive rate of E. vermicularis was 47.2% (359 positives), and sex difference was not remarkable (48.6% in boys and 45.8% in girls). However, the positive rate was the highest in South Dagon (54.6%) followed by Hlaing Thayar (43.8%) and North Dagon (34.8%). This difference was highly correlated with the living standards of the people in each township. Nucleotide sequence of the 5S rDNA from the eggs on the cello-tape (2 children) revealed 99.7% identity with that of E. vermicularis reported in GenBank. The results indicated that E. vermicularis infection is highly prevalent among primary schoolchildren around Yangon, Myanmar.     

Anisakis pegreffii Larvae in Sea Eels (Astroconger myriaster) from the South Sea,Republic of Korea

Anisakis simplex sensu stricto (s.s.), Anisakis pegreffii, Anisakis berlandi (=A. simplex sp. C), and Anisakis typica are the 4 major species of Anisakis type I larvae. In the Republic of Korea (Korea), A. pegreffii, A. berlandi, and A. typica larvae in fish hosts has seldom been documented. In this study, molecular analysis was performed on Anisakis larvae from the sea eels (Astroconger myriaster), the major source of human anisakiasis in Korea, collected from Tongyeong City, a southern coastal area of Korea. All 20 sea eels examined were infected with Anisakis type I larvae (160 larvae; 8 per fish). Their species were analyzed using PCR-RFLP patterns and nucleotide sequences of internal transcribed spacers (ITS1, 5.8 subunit gene, and ITS2) and mitochondrial cytochrome c oxidase 2 (cox2). Most (86.8%; 112/129) of the Anisakis type I larvae were A. pegreffii, and 7.8% (10/129) were A. typica. The remaining 5.4% (7/129) was not identified. Thus, A. pegreffii is the major species of anisakid larvae in sea eels of the southern coast of Korea.     

Plerocercoids of Nybelinia surmenicola (Cestoda: Tentacularidae) in Squids,Todarodes pacificus,from East Sea,the Republic of Korea

A visceral helminth of the squid, Todarodes pacificus, is reported from the East Sea, the Republic of Korea. Total 39 squid samples were purchased from a fish market in Jumunjin-eup, Gangneung-si (City) from August 2014 to July 2015 and were examined for helminth parasites with naked eyes and under a stereomicroscope after opening the abdominal cavity with a pair of scissors. Whitish larval worms were mainly found in the stomach and abdominal cavity of the squid. They were detected in 25 (64.1%) out of 39 squids examined, and the infection density was 7 larvae per infected squid. Spatula-shaped larvae were 8.2×2.0 mm in average size, round to slightly flattened anteriorly, with round hatching posteriorly, and had characteristic 4 tentacles with numerous hooklets in the scolex. The larvae were identified as the plerocercoid stage of Nybelinia surmenicola by their morphological features. This finding represents a new host record and the first report of N. surmenicola infection in T. pacificus squids from the east coast of Korea.     

Sero-epidemiological survey of gnathostomiasis in Lao PDR

While human gnathostomiasis cases have been reported sporadically in Lao PDR since 1975, little is known about the disease in this country. We aimed to investigate sero-prevalence of gnathostomiasis and Gnathostoma species in Lao PDR. One village each in the north, central and south regions of Lao PDR was selected as the study sites. Overall, 125 (29.8%) of 420 sera from the randomly selected participants were sero-positive by immunoblot technique, with anti-Gnathostoma IgG antibody against the 24 kDa fraction. The sero-prevalence was high in the central (47.1%) and south (38.6%), but low (3.6%) in the north. Risk factor analyses revealed that the consumption of raw/undercooked fish was significantly associated with Gnathostoma sero-positivity (95% CI 1.05–17.05, P = 0.042). The sero-positivity significantly increased with the age of the participants. Several fish, swamp eels, and frogs collected from central and southern Lao were infected with G. spinigerum advanced 3rd-stage larvae. Channa limbata (red-tailed snakehead fish) was identified as a natural second intermediate host of G. spinigerum. Eggs of G. spinigerum were found in dog feces collected in the south. Gnathostomiasis is endemic in central and southern Laos, so that preventive measures should be introduced for people living in these regions.     

Cutaneous Gnathostomiasis with Recurrent Migratory Nodule and Persistent Eosinophilia: a Case Report from China

The present study reports a human case of cutaneous gnathostomiasis with recurrent migratory nodule and persistent eosinophilia in China. A 52-year-old woman from Henan Province, central China, presented with recurrent migratory reddish swelling and subcutaneous nodule in the left upper arm and on the back for 3 months. Blood examination showed eosinophila (21.2%), and anti-sparganum antibodies were positive. Skin biopsy of the lesion and histopathological examinations revealed dermal infiltrates of eosinophils but did not show any parasites. Thus, the patient was first diagnosed as sparganosis; however, new migratory swellings occurred after treatment with praziquantel for 3 days. On further inquiring, she recalled having eaten undercooked eels and specific antibodies to the larvae of Gnathostoma spinigerum were detected. The patient was definitely diagnosed as cutaneous gnathostomiasis caused by Gnathostoma sp. and treated with albendazole (1,000 mg/day) for 15 days, and the subsequent papule and blister developed after the treatment. After 1 month, laboratory findings indicated a reduced eosinophil count (3.3%). At her final follow-up 18 months later, the patient had no further symptoms and anti-Gnathostoma antibodies became negative. Conclusively, the present study is the first report on a human case of cutaneous gnathostomiasis in Henan Province, China, based on the past history (eating undercooked eels), clinical manifestations (migratory subcutaneous nodule and persistent eosinophilia), and a serological finding (positive for specific anti-Gnathostoma antibodies).     

Detection of Gnathostoma spinigerum Antibodies in Sera of Non-Traumatic Subarachnoid Hemorrhage Patients in Thailand

Gnathostoma spinigerum can cause subarachnoid hemorrhage (SAH). The detection of specific antibodies in serum against G. spinigerum antigen is helpful for diagnosis of neurognathostomiasis. There is limited data on the frequency of G. spinigerum infection in non-traumatic SAH. A series of patients diagnosed as non-traumatic SAH at the Srinagarind Hospital, Khon Kaen University, Thailand between January 2011 and January 2013 were studied. CT or MR imaging of the brain was used for diagnosis of SAH. Patients were categorized as aneurysmal subarachnoid hemorrhage (A-SAH) or non-aneurysmal subarachnoid hemorrhage (NA-SAH) according to the results of cerebral angiograms. The presence of specific antibodies in serum against 21- or 24-kDa G. spinigerum antigen was determined using the immunoblot technique. The detection rate of antibodies was compared between the 2 groups. Of the 118 non-traumatic SAH patients for whom cerebral angiogram and immunoblot data were available, 80 (67.8%) patients had A-SAH, whereas 38 (32.2%) had NA-SAH. Overall, 23.7% were positive for specific antibodies against 21- and/or 24-kDa G. spinigerum antigen. No significant differences were found in the positive rate of specific antibodies against G. spinigerum in both groups (P-value=0.350).     

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.     

Modulation of Antibody Responses against Gnathostoma spinigerum in Mice Immunized with Crude Antigen Formulated in CpG Oligonucleotide and Montanide ISA720

This study aimed to investigate the antibody responses in mice immunized with Gnathostoma spinigerum crude antigen (GsAg) incorporated with the combined adjuvant, a synthetic oligonucleotide containing unmethylated CpG motif (CpG ODN 1826) and a stable water in oil emulsion (Montanide ISA720). Mice immunized with GsAg and combined adjuvant produced all antibody classes and subclasses to GsAg except IgA. IgG2a/2b/3 but not IgG1 subclasses were enhanced by immunization with CpG ODN 1826 when compared with the control groups immunized with non-CpG ODN and Montanide ISA or only with Montanide ISA, suggesting a biased induction of a Th1-type response by CpG ODN. After challenge infection with live G. spinigerum larvae, the levels of IgG2a/2b/3 antibody subclasses decreased immediately and continuously, while the IgG1 subclass remained at high levels. This also corresponded to a continuous decrease of the IgG2a/IgG1 ratio after infection. Only IgM and IgG1 antibodies, but not IgG2a/2b/3, were significantly produced in adjuvant control groups after infection. These findings suggest that G. spinigerum infection potently induces a Th2-type biased response.     

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.     

A Recombinant Matrix Metalloproteinase Protein from Gnathostoma spinigerum for Serodiagnosis of Neurognathostomiasis

Neurognathostomiasis is a severe form of human gnathostomiasis which can lead to disease and death. Diagnosis of neurognathostomiasis is made presumptively by using clinical manifestations. Immunoblotting, which recognizes antigenic components of molecular mass 21 kDa and 24 kDa in larval extracts of Gnathostoma spinigerum (Gs 21/24), has high sensitivity and specificity for diagnosis of neurognathostomiasis. However, only very small amounts of the Gs 21/24 antigens can be prepared from parasites harvested from natural or experimental animals. To overcome this problem, we recently produced a recombinant matrix metalloproteinase (rMMP) protein from G. spinigerum. In this study, we evaluated this rMMP alongside the Gs 21/24 antigens for serodiagnosis of human neurognathostomiasis. We studied sera from 40 patients from Srinagarind Hospital, Khon Kaen University, Thailand, with clinical criteria consistent with those of neurognathostomiasis, and sera from 30 healthy control adults from Thailand. All sera were tested for specific IgG antibodies against both G. spinigerum crude larval extract and rMMP protein using immunoblot analysis. The sensitivity and specificity for both antigenic preparations were all 100%. These results show that G. spinigerum rMMP protein can be used as an alternative diagnostic antigen, in place of larval extract, for serodiagnosis of neurognathostomiasis.     

Qualitative assessment of the diet of European eel larvae in the Sargasso Sea resolved by DNA barcoding

European eels (Anguilla anguilla) undertake spawning migrations of more than 5000 km from continental Europe and North Africa to frontal zones in the Sargasso Sea. Subsequently, the larval offspring are advected by large-scale eastward ocean currents towards continental waters. However, the Sargasso Sea is oligotrophic, with generally low plankton biomass, and the feeding biology of eel larvae has so far remained a mystery, hampering understanding of this peculiar life history. DNA barcoding of gut contents of 61 genetically identified A. anguilla larvae caught in the Sargasso Sea showed that even the smallest larvae feed on a striking variety of plankton organisms, and that gelatinous zooplankton is of fundamental dietary importance. Hence, the specific plankton composition seems essential for eel larval feeding and growth, suggesting a linkage between eel survival and regional plankton productivity. These novel insights into the prey of Atlantic eels may furthermore facilitate eel larval rearing in aquaculture, which ultimately may replace the unsustainable use of wild-caught glass eels.     

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.     

Comparative Study of Antibiofilm Activity of Copper Oxide and Iron Oxide Nanoparticles Against Multidrug Resistant Biofilm Forming Uropathogens

The effectiveness of the metal oxide nanoparticles viz. CuO and Fe₂O₃ as antibacterial agents against multidrug resistant biofilm forming bacteria was evaluated. CuO nanoparticles were also experimented for antibiofilm and time kill assay. The CuO displayed maximum antibacterial activity with zone of inhibition of (22 ± 1) mm against methicillin resistant Staphylococcus aureus (MRSA) followed by Escherichia coli (18 ± 1) mm. The Fe₂O₃ showed the zone of inhibition against MRSA of (14 ± 1) mm followed by E. coli (12 ± 1) mm. CuO proved to be more toxic than Fe₂O₃ nanoparticles showing significantly high antibacterial activity and found to possess dose dependent antibiofilm properties.     

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.     

Will krill fare well under Southern Ocean acidification?

Antarctic krill embryos and larvae were experimentally exposed to 380 (control), 1000 and 2000 µatm pCO₂ in order to assess the possible impact of ocean acidification on early development of krill. No significant effects were detected on embryonic development or larval behaviour at 1000 µatm pCO₂; however, at 2000 µatm pCO₂ development was disrupted before gastrulation in 90 per cent of embryos, and no larvae hatched successfully. Our model projections demonstrated that Southern Ocean sea water pCO₂ could rise up to 1400 µatm in krill''s depth range under the IPCC IS92a scenario by the year 2100 (atmospheric pCO₂ 788 µatm). These results point out the urgent need for understanding the pCO₂-response relationship for krill developmental and later stages, in order to predict the possible fate of this key species in the Southern Ocean.     

Nematode Electrocution

The effects of electric shock on Panagrellus redivivus adults and larvae and Meloidogyne incognita acrita larvae were studied. The nematodes were placed in tap water between two stainless steel electrodes, spaced 2 mm apart and cemented to a glass slide. Electric potentials of 1, 5, 10, 15, 20, 30, and 60 vdc/mm and vac/mm were applied for periods of 1 sec to 5 rain at 0.05 to 77 ma. The results demonstrated that ac or dc electric shocks as low as 5 v/mm for larvae and 10 v/mm for adults can be lethal. Some larvae and eggs within the body of P. redivivus females were not affected at 600 v/ram. Potentials of 20 and 60 vdc/mm for 2-sec stimulated hatch of Meloidogyne eggs.