Advanced Third-stage Larvae Research Articles

Abnormalities on cephalic hooklets of advanced third-stage larvae from Gnathostoma Owen, 1836 (Nematoda: Gnathostomidae) collected from Mexican rivulus Millerichthys robustus Costa, 1995 (Ciprinodontiformes: Rivulidae) in Tlacotalpan, Veracruz, Mexico

Morphological abnormalities were observed on the cephalic bulb hooklets of advanced third-stage larvae (AdvL(3)) of genus Gnathostoma. The larvae were obtained from the fish "Mexican rivulus" Millerichthys robustus collected from a seasonal pond near Tlacotalpan, Veracruz, Mexico. The abnormalities involved (1) extra rudimentary hooklets, located between the four rows and after the fourth row, (2) branched or lobulated hooklets, and (3) fragmented hooklets not uniformly disposed in rows. The alterations observed on the cephalic bulb hooklets do not represent intraspecific variations, and they may be considered as a potential tool for assessing the presence of pollutants or stressors located within the ecosystem.

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.

Identification and characterization of a cathepsin L-like cysteine protease from Gnathostoma spinigerum

Gnathostoma spinigerum is a causative agent of human gnathostomiasis, a common parasitic disease involving skin and visceral organs, especially the central nervous system. In this study, we identified a cDNA encoding a cathepsin L-like cysteine protease (GsCL1) from the λZAP cDNA library of G. spinigerum advanced third-stage larva (aL3) and characterized the biochemical properties of the recombinant enzyme. The cloned cDNA of 1484bp encoded 398 amino acids which contained a typical signal peptide sequence (23 amino acids), a pro-domain (156 amino acids), and a mature domain (219 amino acids) with an approximate molecular weight of 24kDa. The deduced amino acid sequence of GsCL1 gene showed 53–64% identity to cathepsin L proteases of various organisms including a cathepsin L family member (cpl-1) of Caenorhabditis elegans. Recombinant proGsCL1 expressed in Pichia pastoris showed typical biochemical characteristics of cysteine proteases. The expressed enzyme displayed optimal protease activity toward Z-Phe-Arg-AMC substrate at pH 6.0 but not toward Z-Arg-Arg-AMC. The activity was sensitive to cysteine protease inhibitors E-64 and K11777. The preference for large hydrophilic and aromatic residues in the P2 position (I, L, F, W, U, V) was typical of cathepsin L proteases. Mouse anti-GST-proGsCL1 serum showed reactivity with 35-, 38- and 45-kDa proteins in the aL3 extracts. These proteins were shown to localize inside the intestinal cells of aL3.

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.

Evaluation of human IgG class and subclass antibodies to a 24 kDa antigenic component of Gnathostoma spinigerum for the serodiagnosis of gnathostomiasis

The immunoglobulin G class (total IgG) and subclass (IgG1, IgG2, IgG3, and IgG4) antibody responses to the spirurid worm Gnathostoma spinigerum were analyzed by immunoblotting technique for the antibodies' potential use in the serodiagnosis of human gnathostomiasis. Serum samples from patients with proven gnathostomiasis and from clinically suspected cases of gnathostomiasis with migratory swelling were tested. Sera from patients with other parasitic illnesses and from healthy volunteers were also analyzed. The total IgG antibody to an antigenic band of approximately 24 kDa from a somatic extract of G. spinigerum advanced third-stage larvae (GS24) gave the highest sensitivity (91.6%) and 87.8% specificity. Individual IgG subclass detection had a lower sensitivity than the detection of specific total IgG antibody, but IgG4 had a slightly higher specificity (93.9%). However, for cost effectiveness, we suggest that anti GS24 total IgG is sufficient for the routine serodiagnosis of human gnathostomiasis as well as for seroepidemiological studies in developing countries.

INTRAVITREAL GNATHOSTOMIASIS AND REVIEW OF LITERATURE

Intravitreal gnathostomiasis is a rare disease. To our knowledge, only five case reports are available in the literature wherein the parasites were surgically removed from the vitreous cavity and the species were identified. In this report, we document the clinical features of intravitreal gnathostomiasis along with the route of entry and parasitic migration pattern in ocular tissue, surgical management, and electron microscopic features of the retrieved advanced third-stage larva of Gnathostoma spinigerum (aL3) for a 48-year-old Indian woman with excellent postsurgical visual recovery.

Protein profile analysis from advanced third-stage larvae (AdvL3) and adult worms of Gnathostoma binucleatum (Nematoda: Spirurida)

Proteins from crude extracts of advanced third-stage larvae and adult Gnathostoma binucleatum nematode worms showed protein profiles in SDS-PAGE analysis similar to Echinococcus granulosus, Trichinella spiralis, Dipylidium caninum, Ancylostoma caninum, Ascaris lumbricoides and Toxocara canis. The immunoblot analysis of the human serum infected or suspected to be infected with G. binucleatum using the total larvae extract recognized the 40, 60, 80 and 115 kDa proteins and using the total adult worm extract recognized only the 80 and 115 kDa proteins. However, the 115 kDa protein showed cross-reactions with A. caninum, A. lumbricoides, T. canis and D. caninum with human serum positive to gnathostomosis, while the 40 kDa protein was only recognized with the G. binucleatum total larvae extract. The results obtained suggest that the use of antigens from the advanced third-stage larvae of the parasite were best recognized for immunodiagnosis of gnathostomosis.

Diagnostic values of IgG4 in human gnathostomiasis

The diagnostic values of immunoglobulin G subclass antibodies from patients with gnathostomiasis were assessed by immunoblot technique. Antigen was prepared from crude extracts of Gnathostoma spinigerum advanced third-stage larvae obtained from naturally infected eels. The sera were obtained from 14 parasite-confirmed gnathostomiasis cases, 63 patients with other helminthic infections and 13 healthy controls. Nine prominent IgG4 reactive bands appeared with molecular weights of 94, 51, 47, 43, 38, 24, 21, 20 and 15 kDa. The diagnostic sensitivity of each of the nine reactive bands ranged from 100% to 64.3% in 14 parasite-confirmed gnathostomiasis cases. All (100%) confirmed cases recognized the 21 kDa antigenic band, but not other helminthic infections or parasite-free control. Recognition of 21 kDa antigen in G. spinigerum advanced third-stage larvae crude extracts is the most specific diagnostic marker for human gnathostomiasis, with 100% sensitivity and specificity. The 20 and 24 kDa protein bands were additional diagnostic bands for confirming diagnosis of infection where the 21 kDa band was faint. No specific binding of IgG1, IgG2, or IgG3 antibodies was observed in any sera from confirmed gnathostomiasis cases.

Confirmation of Gnathostoma binucleatum Almeyda-Artigas, 1991, Advanced Third-Stage Larvae in Tres Palos Lagoon, Mexico, by Morphological and Molecular Data

Advanced third-stage larvae of Gnathostoma sp. corresponding to 6 morphotypes, distinguished on the basis of the shape of the cephalic hooklets, were collected from the muscle tissue of 5 species of freshwater fish (i.e., Dormitator latifrons, Eleotris picta, Gobiomorus maculatus, Ariopsis guatemalensis, and Cichlasoma trimaculatum) in Tres Palos Lagoon, Guerrero, Mexico. Principal components analysis of 4 morphological characters cluster all samples in a single taxonomic group. A minimum amount of variation was observed among ITS2 sequences of 3 morphotypes and Gnathostoma binucleatum obtained from GenBank (0-0.84%). The observed variation among morphotypes 1, 2, and 3 is the result of intraspecific variability of G. binucleatum supported by morphology and DNA. Morphotypes 4, 5, and 6 belong to the same taxon on the basis of morphology of the hooklets only. For an accurate morphological diagnosis of the causative agent of gnathostomiasis, it is necessary to develop similar studies with other species of the genus.

Gnathostoma binucleatum: Excretion–secretion antigen analysis obtained from advanced third-stage larvae in in vitro culture

The paper describes an introductory characterization of antigenic stimulation of excretion–secretion products (ESP) of Gnathostoma binucleatum advanced third-stage larvae cultured in vitro and proteinases present in this products. Excretory and secretory proteins were obtained after 10 larvae were maintained in 5% CO 2 RPMI medium. The supernatant was collected each week for two months. The proteins were dialyzed, concentrated, and separated in 10% SDS–PAGE gels under reducing conditions and transferred to nitrocellulose paper for immunoblot analyses. G. binucleatum immunized mice serum was used to determine protein antigenicity. Four proteins of 40, 80, 120, and 208 kDa persisted for two months and three proteins, 80, 120, and 208 kDa were recognized for antibodies of mice. In SDS–PAGE gelatin substrate gels ESP resolved as two proteins with molecular weight of 80 and 208 kDa that were sensitive to a metalloproteinase inhibitor, and thus it may be inferred that they might be used for diagnosis of gnathostomiasis.

Surface ultrastructure of the advanced third-stage larvae of Gnathostoma nipponicum.

The surface ultrastructure of advanced third-stage larvae (AL3) of Gnathostoma nipponicum was studied using scanning electron microscopy. The larvae were recovered from the grass snake Rhabdophis tigrina in the Republic of Korea. Parasites had a globular head bulb with a pair of lips at the anterior end and 2 labial papillae and an amphid on each lip. The head bulb was characteristically armed with 3 transverse rows of hooklets, averaging 36, 38, and 43 in number, increasing posteriorly. A total of 213-232 minute unidentate cuticular spines were present along the entire length of the larvae, forming the transverse striations. Two pairs of cervical papillae were located between the 8th and 12th transverse striations, and a pair of body papillae was seen laterally on the posterior third of the body. A pair of caudal phasmids was recognized near the posterior extremity. The surface ultrastructure of AL3 of G. nipponicum is unique compared with that of other species.

AN OUTBREAK OF GNATHOSTOMIASIS AMONG KOREAN EMIGRANTS IN MYANMAR

Thirty-eight (designated as cases) of 60 Korean emigrants who consumed raw fresh water fish in Yangon, Myanmar developed migratory swellings and creeping eruptions on the back, abdomen, flank, and other cutaneous areas 1-10 weeks later. The symptoms included itching, nodule formation, fatigue, urticaria, fever, pain on the skin, and erythematous plaques. Skin biopsies of two cases revealed no parasites. However, the mean +/- SD peripheral blood eosinophilia among the cases was 6.3 +/- 6.5% (n = 29) and 9.0 +/- 9.8% (n = 26) in two examinations. An enzyme-linked immunosorbent assay of their serum samples, using Gnathostoma doloresi adult worms as the antigen, showed mean +/- SD optical densities of 0.47 +/- 0.29 (n = 28) and 0.32 +/- 0.20 (n = 30) in two examinations and 0.12 +/- 0.09 (n = 50) in healthy controls. Two advanced third-stage larvae of G. spinigerum were found in two of six catfish purchased at a local market in Yangon. The outbreak of the human infection is suggested to have been due to G. spinigerum, which is known to live out its life cycle in the Yangon area of Myanmar.

Acute outbreak of gnathostomiasis in a fishing community in Sinaloa, Mexico

An increasing number of human cases of gnathostomiasis have been reported in Sinaloa, Mexico, most of whom have a custom of eating of raw fish dishes such as ‘cebiche’. Here we report five adult patients, three women and two men, having an acute episode of vomiting and abdominal pain a few minutes after eating a dish of cebiche prepared from a spotted sleeper perch ( Eleotris picta) fished from a nearby lake in southern Sinaloa. All five patients experienced acute throat pain, chest and joint pains, headache and fever. One patient, a 55-year-old male, was hospitalized with suspected pancreatitis and pneumonia. By 8–9 days later, all five patients developed between three and 12 edematous, migrating skin lesions on the back, abdomen, upper and lower extremities, face, eye and scalp. By ELISA, all of them were sero-positive to Gnathostoma doloresi antigen and had elevated IgE levels. Eosinophilia was found in two patients. These patients lived in an agricultural and fishing community. In this community we carried out a sero-epidemiological survey and study of living conditions in a random sample of 309 individuals distributed in 74 households. Frequent consumption of raw fish was reported in 36% of households, and 12 individuals had a clinical history of migrating skin lesions. The sero-prevalence to Gnathostoma antigens was 34.95%. Five fish species and four species of ichthyophagous birds collected from three lakes in the village and a nearby estuary were infected with the advanced third-stage larvae of G. binucleatum, a species found in Ecuador and Mexico. The results describe the first known outbreak of acute gnathostomiasis on the American continent.

Characterization of Monoclonal Antibodies Against Gnathostoma nipponicum

Monoclonal antibodies (mAbs) were produced against the proteins of advanced third-stage larvae (AdL3) of Gnathostoma nipponicum. Six mAbs (Gn2C3, Gn2H3, Gn4C3, Gn4E9, GnSH1, and Gn10B7) were obtained as determined by enzyme-linked immunosorbent assay (ELISA). Gn4E9 and GnSH1 seemed to be genus-specific, as they did not cross-react with Anisakis sp., Dirofilaria immitis, Gongylonema pulchrum, Toxocara canis, Trichinella sp., Trichuris vulpis, Metagonimus sp., or Spirometra erinaceieuropaei by ELISA. Immunohistochemistry showed that Gn2C3, Gn4E9, and Gn5H1 reacted strongly with the central esophagus; Gn2H3 reacted with cuticle,muscle, intestine, and the cervical sac; and Gn4C3 and Gn10B7 reacted with cuticle, muscle, esophagus, intestine, and the cervical sac of AdL3. In Western blotting analysis, Gn2C3, Gn4E9, and Gn5H1 reacted to 60-, 53-, 46-, and 41-kDa proteins; Gn4C3 reacted to the AdL3 protein of G. nipponicum (>42 kDa). Moreover, proteins purified using a mAb Gn4E9 immunoprecipitation method (sizes 60-, 53-, 46-, and 41-kDa) were used as antigens in ELISAs. A significant difference (P < 0.01) was shown between mouse sera infected with G. nipponicum and sera infected with Trichnella sp. or not infected. These results provide a rationale for evaluating esophageal proteins for the development of diagnostic methods for detecting G. nipponicum or Gnathostoma sp. infections.

Parasitic Helminths Collected from the Japanese Giant Salamander, Andrias japonicus (Temminck, 1837) (Amphibia: Cryptobranchidae), in Japan

Helminth parasites collected at postmortem from the alimentary tracts of 5 Japanese giant salamanders, Andrias japonicus (Temminck, 1837), are reported. One trematode, Liolope copulans (Cohn, 1902); and 5 nematodes, Amphibiocapillaria tritonispunctati (Diesing, 1851), Dioctophyme renale (Goeze, 1782) (larva), Spiroxys hanzaki Hasegawa, Miyata et Doi, 1998, Kathlaniidae sp. (larva), and Physalopteroidea sp. (larva) were found. Examination of premolt and molting larvae of S. hanzaki showed that the advanced third-stage larvae molt directly to the adult stage. Strict host specificity of some helminths is surmised to be linked to the piscivorous nature of the giant salamander. This is the first report of A. tritonispunctati and D. renale from A. japonicus. The chromosome number of L. copulans is recorded for the first time.

Molecular cloning of a gene encoding matrix metalloproteinase-like protein from Gnathostoma spinigerum.

The advanced third-stage larvae (aL3) of Gnathostoma spinigerum contain a 24 kDa glycoprotein with diagnostic potential. Immunoscreening with the monoclonal antibody to the 24-kDa protein (mAb GN6/ 24) has identified a cDNA clone with an insert of 932 base pairs (bp). The insert contains a full-length gene of 732 bp encoding a protein that is 33-39% similar to matrix metalloproteinases (MMPs) of Caenorhabditis elegans and several lower and higher vertebrates. The MMP-like protein of G. spinigerum possesses the catalytic domain, but lacks the propeptide and hemopexin-like domains found in other MMPs. A signal peptide of 23 amino acids at its amino terminus indicates that it is a secretory protein, which is confirmed by Western blot analysis showing the presence of the 24 kDa protein in the excretory-secretory products of aL3.

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.

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.

Clinical manifestations and immunodiagnosis of gnathostomiasis in Culiacan, Mexico.

Gnathostomiasis was first described in Mexico in 1970, and endemic areas have been spreading in six states of this country. In Culiacan, Sinaloa, 300 cases of cutaneous larva migrans were recorded between January 1992 and December 1995. In addition, a Gnathostoma larva was surgically removed from the eye of one patient. Cutaneous lesions were observed mainly on the face, neck, arms, and legs. About 70% of the patients showed eosinophilia. A skin biopsy was carried out on 35 patients and the parasite was identified in histopathologic sections of 12 of these patients. In four patients, the larva migrated out spontaneously from the skin. An enzyme-linked immunosorbent assay using a crude somatic extract of adult Gnathostoma doloresi worms showed that 93% of the patients were seropositive, confirming the reliability of clinical diagnosis. A total of 14 advanced third-stage Gnathostoma larvae were found in four species of ichthyophagous birds captured on dams and dikes near the city of Culiacan. Scanning electron micrographs of human and bird larvae showed that they were morphologically indistinguishable from G. spinigerm. We conclude that the life cycle of Gnathostoma has been established in Sinaloa, and has become a serious public health issue for residents.

Human gnathostomiasis caused by Gnathostoma doloresi, with particular reference to the parasitological investigation of the causative agent

Gnathostomiasis caused by Gnathostoma doloresi Tubangui was observed in a 46-year-old Japanese man, a clerk, who resided in the Fukuoka prefecture of Kyushu district, in southern Japan. The first symptom noted by the patient was epigastric pain, which occurred 3 days after he had consumed the raw flesh of a brook trout, Oncorhynchus masou masou (Brevoort), from a trout farm in neighbouring Kumamoto prefecture. Three days later, he developed creeping eruptions on his trunk. The eruptions spread and the patient was admitted to hospital 18 days after eating the trout. No nematode larvae were found in any of four skin biopsies collected on admission. However, vesicles appeared on the lower abdomen of the patient 20 days after he had eaten the trout, and a small nematode was removed from one of them. One day after the nematode had been excised, a cutaneous swelling suddenly occurred on the patient's lower left jowl and this persisted for a week. All the skin abnormalities gradually decreased from day 25 post-ingestion and had disappeared by day 30. The isolated nematode was identified as an advanced third-stage larva of G. doloresi, based on its morphology and the number of hooklets on its head-bulb.