Towards a suitable antigen for diagnosis of Gnathostoma spinigerum infection.

, , , and 1992. Towards a suitable antigen for diagnosis of Gnathostoma spinigerum infection. International Journal for Parasitology 22: 1151–1156. Advanced third-stage larvae of G. spinigerum were obtained from two separate sources, namely from cysts in the livers of naturally infected eels (L3E) and from experimentally infected mice (L3M). Morphology of the L3E was studied microscopically. The larvae were homogenized in distilled water, 1% Triton X-100 or 1% sodium deoxycholate containing protease inhibitors. Protein compositions of the three crude extracts were compared, on the same weight basis, by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie brilliant blue staining while their antigenicities were studied by Western blot analysis using serum of a patient with parasitologically confirmed gnathostomiasis. Distilled water was found to be the best extraction solution in solubilizing proteins especially the diagnostic antigen, namely the 24,000 (24 kDa) mol. wt component from the larvae. The L3E and L3M contained relatively equal amounts of the 24 kDa antigen. This diagnostic component was anatomically located in the body fluid, oesophagus and intestine of the larva.     

Monoclonal antibody to a diagnostic Mr 24,000 antigen of Gnathostoma spinigerum.

Crude water extract (CA) was prepared from the advanced third-stage larvae of Gnathostoma spinigerum collected from livers of naturally infected eels. The extract was partially purified by chromatofocussing column chromatography and the fraction which contained specific antigen of G. spinigerum which was an Mr 24,000 glycoprotein was used to immunize five Balb/c mice for preparing immune splenocytes. Spleen cells were collected from one mouse which showed high serum titre by indirect enzyme-linked immunosorbent assay and contained specific antibody to the Mr 24,000 antigen as checked by Western blot analysis. The spleen cells were fused with myeloma Sp2/0 cells at a ratio of 10 spleen cells per one myeloma cell using polyethylene glycol 3350 as a fusogen. Thirteen out of 174 growing polyclones (7.5%) produced antibodies to the partially purified CA fraction. Among them, two polyclones produced antibody directed to the Mr 24,000 protein. These two polyclones were subjected to monocloning by limiting dilution and a monoclone GN6/24 which produced monoclonal antibody to the specific Mr 24,000 protein of G. spinigerum was obtained.     

Purification of Gnathostoma spinigerum specific antigen and immunodiagnosis of human gnathostomiasis.

Specific antigen of G. spinigerum which has been shown to be a protein with a relative mol. wt of 24,000 (24K) was prepared from the advanced third-stage larvae (L3) obtained from the livers of naturally infected eels. The L3 were ground and extracted with water. Purification procedures involved gel filtration, chromatofocussing and anion exchange column chromatographies, while characterization of the specific antigen was performed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and staining, Western blot analysis and isoelectric focussing. The specific antigen which has a pI of 8.5 was used as antigen in the indirect enzyme-linked immunosorbent assay (ELISA) to detect specific antibody in four groups of individuals, namely five parasitologically diagnosed gnathostomiasis patients (group 1); 15 clinically diagnosed gnathostomiasis patients (group 2); 136 patients with other parasitic infections (group 3); and 25 normal healthy parasite-free controls. Sensitivity, specificity and predictive values (positive and negative) of the assay were 100%.     

Specific antigen of Gnathostoma spinigerum for immunodiagnosis of human gnathostomiasis

Sera from four patients with parasitologically confirmed gnathostomiasis, 15 patients with presumptive gnathostomiasis, 64 patients with various parasitic infections and 19 healthy adults were studied by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis for their reactivities against somatic extract of Gnathostoma spinigerum third-stage larvae (L3). It was found that the L3 extract was highly complex consisting of more than 20 antigenic components, a few of which gave reactions with sera from the healthy controls. Extensive cross-reactions of the parasite's antigen with sera from patients with other parasitic infections occurred. A specific antigen of G. spinigerum with a mol. wt of 24,000 (24k) was found to react with all parasitologically proven patients, five of the presumptive patients, one of the patients with other parasitic infections and none of the healthy individuals. This 24k component of G. spinigerum is a potential diagnostic antigen for use in the immunodiagnosis of human gnathostomiasis.     

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.     

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).     

Seasonal variation in the prevalence and intensity of canine Gnathostoma spinigerum infection in northeastern Thailand.

Gnathostoma spinigerum was found in gastric nodules in 4.1% of 2940 dogs surveyed in northeastern Thailand. The prevalence and worm burden of G. spinigerum exhibited a seasonal fluctuation. The parasites were more abundant in the rainy season and the early winter (August-December) than in the summer (April-March). Most parasites were sexually mature between August and December while immature worms were observed during March and April. The distribution of gnathostomes within the sampled dogs was highly dispersed and few animals were found to harbour more than five worms.     

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.     

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.     

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.     

Recognition of deglycosylated larval proteins of Gnathostoma spinigerum by a monoclonal antibody and human gnathostomiasis antiserum.

The study on the recognition of 35S-labelled somatic antigens of Gnathostoma spinigerum advanced third-stage larva (aL3) has revealed that the mAb GN6/24 immunoprecipitated 26- and 24-kDa proteins from the undigested and N-glycosidase F-digested larval extracts, respectively. The recognition of the deglycosylated form of the glycoprotein indicated that the mAb reacted with the peptide epitope on the 26-kDa protein. Human gnathostomiasis antiserum immunoprecipitated most of the N-glycosidase F-digested larval proteins including the deglycosylated 26-kDa protein.     

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.     

Analysis of sequence variation in Gnathostoma spinigerum mitochondrial DNA by single-strand conformation polymorphism analysis and DNA sequence

Morphological variations were observed in the advance third stage larvae of Gnathostoma spinigerum collected from swamp eel (Fluta alba), the second intermediate host. Larvae with typical and three atypical types were chosen for partial cytochrome c oxidase subunit I (COI) gene sequence analysis. A 450 bp polymerase chain reaction product of the COI gene was amplified from mitochondrial DNA. The variations were analyzed by single-strand conformation polymorphism and DNA sequencing. The nucleotide variations of the COI gene in the four types of larvae indicated the presence of an intra-specific variation of mitochondrial DNA in the G. spinigerum population.     

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.     

Scanning electron microscopy of the cuticular armature of the nematode Gnathostoma spinigerum Owen, 1836 from cats in Laos

The body surface of an immature female Gnathostoma spinigerum found for the first time in the definitive host (Felis catus f. domestica) in Laos was studied using a scanning electron microscope. All types of cuticular spines, which are one of the most important features for species identification of gnathostomid nematodes, together with their spatial arrangement, are described and figured.     

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.     

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.     

Efficacy of mebendazole and albendazole against Trichinella spiralis in mice

Changes in the sensitivity of Trichinella spiralis to anthelmintic treatment during the first 3 days of infection in mice were studied. Oral administration of either mebendazole or albendazole at 6.25 mh/kg 2 hr after exposure to infection eliminated 95-100% of the worms as determined at necropsy on day 7 postinoculation. Beyond the first day of infection the sensitivity of the parasite to benzimidazole therapy was much reduced and an oral dose of 50 mg/kg was only partially but significantly active against the adult worms. Despite decline in drug sensitivity during the enteral phase, gavage administration of either mebendazole or albendazole at 50 mg/kg for 5 consecutive days during the invasive phase of infection significantly reduced (96 and 67%, respectively) the number of larvae subsequently recovered from host musculature on day 45 postinoculation.