Surface Ultrastructure of Larvae and Adults of Gnathostoma binucleatum obtained in Mexico

Surface Ultrastructure of Larvae and Adults of Gnathostoma binucleatum obtained in Mexico Masataka Koga 1), Hiroshige Akahane 2), Rafael Lamothe-Argumedo 3), David Osorio-Sarabia 3), Luis García-Prieto3), Juan Manuel Martinez-Cruz 4), Sylvia paz Diaz Camacho 5) 1) Department oí Parasitology, Faculty oí Medicine, Kyushu. University, Fukuoka 812-8582, ]apan 2) Department oí Parasitology, School oí Medicine, Fukuoka. University, Fukuoka 814-0180, ]apan 3) Laboratorio de Helmintologia, Departament de Zoología. Instituto de Biologia, Universidad Nacional Autonoma de Mexico D. F., Mexico 4) Pedro Garcia No. 918 Tierra Blanca, Veracruz, Mexico 5) Facaltad de Ciencias Quimico-Biologicas, Universidad. Autonoma de Sinaloa, Culiacan, Sinaloa, Mexico Abstract: We examined the morphology of gnathostome larvae obtained in Temazcal, Sinaloa and Tepic Mexico, mainly using scanning electron microscopy (SEM). The larvae from Temazcal further experimentally infected two dogs and 8-9 months later. We found adults of those larvae in the stomachs of them. They are al so examined the surface morphology by SEM. Upon the larvae obtained from the muscles oí fishes and pelicans, the average body length was 4. 67 mm. The head had four transverse rows of hooklets, and average number of each row was 39.3, 43.9, 46.6 and 49.8 respectively. The bodies were wholly covered with minute cuticular spines along their transverse striations. The average number of striations varied from 227-275. The cervical papillae were situated between the 13th-17th transverse striations. An excretory pore also located ventrally between the 24th-28th transverse striations. On the other hand, the morphology of the adult worms was examined by SEM. The adults demonstrated tridentate cuticular spines in the anterior forefront regions. The shape of the cuticular spines changed to di- and monodentate forms in the anterior one-third of the body. Very minute monodentate spines covered the posterior two-thirds of the body. The ventral surface of the male tail had 4 pairs of caudal papillae and 3 pairs of small papillae. The spines in this area were short. We found no pits on the eggshell surface of our specimens. The three previously reported indigenous to Latin America, G.turgidum, G.procyonis, and G.americanum, have many pits on their eggshells. The present species was more similar to G.binucleatum (G.spinigerum). Introduction Gnathostomiasis is an important parasitic zoonosis, which is mainly endemic in Asia so faro In such countries as ]apan, Thailand and Vietnam, people often eat raw fresh water fish. For this reason, this food-borne disease was thought to be limited to Southeast Asian countries for the past 50 years. In 1970, however, a case of human gnathostomiasis was first reported in Mexico (Pelaez and Perez-Reyes, 1970). This patient was neither a traveler nor an immigrant from Southeast Asia. After that, the number of reported gnathostomiasis patients increased drastically and up to the present time, more than 1,000 cases have been diagnosed in Mexico. The endemic area in Mexico includes six states, which are roughly divided into three regions including the Pacific coast (Culiacan), Atlantic coast areas (Tampico) and adjacent regions (Veracruz) of north American country (Ogata et al. 1998). Previously, Lamothe-Argumedo et al. (1989) and Armeyda-Artigas (1991) examined the morphology of Gnathostoma larvae from fish in Oaxaca-Veracruz and later Akahane et al. (1994) al so examined the morphology of the larvae collected from Pelicans in the same are a by light microscopy. We herein report the morphology of Gnathostoma specimens which are considered to be the same species reported from human in Mexico. Larvae from fish and pelicans, and adults recovered from dogs which had infected experimentally with the larvae were examined mainly using scanning electron microscopy. Materials and Methods We collected 3 pelicans from the Presidente Miguel Aleman Reservoir in Temazcal and examined their muscles to collect gnathostome larvae. The muscles were removed, chopped into small pieces, and the pieces cut into thin slices. The slices were then placed between 2 glass plates (10 by 10cm), pressed by hands, and examined under a dissecting microscope. The inspected muscle remnants were next digested in artificial gastric juice (0.2g pepsin in 0.7ml HCl/100ml distilled water) for 3 hours at 37 C to harvest any larvae that had been overlooked. We eventually found a 1

total of 570 lavae. We also examined a natural second intermediate fish host to collect the larvae, especially in Culiacan and Tepic. Two dogs were each infected experimentally with 20 larvae obtained from pelicans, and thereafter, they were maintained at an animal center. To assess egg shedding, fecal examinations were performed once a month starting at the fifth month postinfection. Gnathostoma eggs were first observed in the feces 8 and 9 months after infection. The dogs were then anesthetized by sodium barbital and killed by bleeding from the cervical arteries. The peritonea were then gently opened, and the stomachs were removed and opened by cutting along the lesser curvature. A single hard nodule was evident in the mucous membrane in each stomach. In the nodules from each dogs, 4 (1, 3 ) and 9 (6,3 ) adult worms were obtained. The eggs were moved from the uteri of the gravid female worms for further examination. First the larvae were processed for morphological observations by both light and scanning electron microscopy. Paraffin section specimens were prepared by the conventional methods and stained H E. For the SEM specimen preparations, ten viable larvae from Temazcal and Tepic, and three from Culiacan were washed in distilled water and stored in a refrigerator until the worms died naturally and stretched out completely. They were then fixed in 10% formalin for 7 days. Next, the larvae were washed in running tap water overnight to remove the fixative, and then were transferred to distilled water. The specimens were rinsed twice in Millonig's phosphate buffer and postfixed overnight in 1 % OS04 in the same buffer, as a result bodies became rigid. AlI specimens were then carefully and gradually dehydrated in an ascending series of ethanol, since such specimens often shrink or have surface wrinkles due to rapid dehydration. They were then transferred into amyle acetate, and COz critical-point dried with a Hitachi HCP2 dryer. The specimens were sputter coated with gold and examined with a leol lsm-u3 SEM operated at 15KV. Viable male and female worms and eggs were processed same procedures for preparing SEM specimens. Results As many as 570 larvae were thus finally obtained from three Pelicans in Temazcal.Almost same number of larvae were obtained from about 200 fish in Tepic. On the other hand, only three larvae were found in five egrets in Culiacan. The average body length (lo larvae) of these Mexican larvae was 4.67 mm measured in a relaxed state after nutural death in cold distilled water. The head had four transverse rows of hooklets (Fig. 1), the average number (10 larvae) of each rows was 39.3, 43.9, 46.6 and 49.8 respectively. The typical hooks on the head bulb had sharp tapering points composed of hard keratin that emerged from an oblongate chitinous base (Fig. 2). The body were entirely covered with minute cuticular spines along their transverse striations. The average number (lo larvae) of striations varied from 227 to 275. A pair of cervical papillae was laterally situated between the 13th-17th transverse striations (Fig. 3). In most specimens, the papillae were located between 14th-15th striations. An excretory pore was observed ventrally and the location was between 24th-28th transverse striations (Fig. 4). On the ventral surface of terminal end, a wide anal opening (AP) was visible and transverse striations were spaced at this openings (Fig. 5). The extremity of the larva had a pair of phasmidial pores (PH) which were late rally located (Fig. 6). The morphological features of intestinal cells were of multiple nuclei in the larvae from Temazcal (Fig. 7). The larvae from Sinaloa also had 2-7 nuclei in each intestinal cell (Fig. 8). The either dogs infected the mexican larvae collected in Temazcal had adult worms in their stomach nodules (Fig. 9). The body length of them were 2-2.5 cm in female and 1. 0-1. 5 cm in maleo The adult worm specimens had a hemispherical head bulb armed with 8-9 transverse rows of cephalic hooks (Fig. 10). The body spines irnmediately behind the cephalic bulb were tridentate (Fig.l1) and not multidigitated. One pair of cervical papillae was located laterally near the twentieth transverse striations and had a mammiform shape (Fig. 12). The shapes of the spines around these papillae were mixed, with 2-3 denticles, and only rare unidentate spines. A domelike excretory pore was situated ventrally a little behind the cervical papillae and was covered only by single spines (Fig. 13). These unidentate spines gradually decreased in size posteriorly along the body (Fig. 14), and most posterior spines were minute (Fig. 15). At the posterior half of the body, very minute spines were sparsely evident as less as those in the Thai specimens of G.spinigerum. On the ventral side of the tail of the mail, single-toothed unidentate spines were densely distributed only over the entire extremity. Four pairs of caudal papillae and a few small papillae, which bore no spines were also seen on this side (Fig. 16). The fertilized uterine eggs of this Gnathostoma species were oval (66!: 2.92 by 40!: 3. 1J.1.IIl) and had operculum on one end. The eggshell surface was plain and without pits (Fig.17). This plain, nonpitted appearance is characteristic of this species and is major feature differentiating from G.spinigerum. Discussion Lamothe-Argumedo et al. (1989) described their larval Gnathostoma specimens obtained from Temazcal as Gnathostoma sp. alone. However, based on our observations, his specimens seemed to be the same as those reported by Almeyda-Artigas (1991). Because both specimens of larvae were from fish and waterfowls in the same endemic area of human gnathostomiasis and the descriptions of larval morphology were quite similar. We thus identify this specimen as G.binucleatum (Almeyda-Artigas, 1991) in Table 1. Lamothe-Argumedo et al. (1989) had previously observed larvae 2

from Oaxace, Temazcal, Mexico by SEM. We think that their SEM observations were somewhat insufficient, especially regarding the location of excretory pares and number of transverse striations on larval bodies. We thus re-examined the Temazcal specimens using SEM and discovered some new findings. Furthermore, we also examined the surface structures of the specimens from Sinaloa, Culiacan and Tepic, Nayarit. Previously, five specimens from Sinaloa were observed by Carnacho et al. (1998) using SEM. They mentioned the number of hooklets of four rows on the head buld as 39, 42, 44 and 49 respectively. Furthermore, they recognized one pair of cervical papillae located between the 13th- 15th striation of the cuticular spines on a single larva. The number of transverse striations on the body was more than 200. There were no descriptions regarding the location of the excretory pore. The situations of cervical papillae, excretory pore and the number of transverse striations are very important for the identification of Gnathostoma species at larval stage. As shown in Table 1, the number of transverse striations is even more than 200 in G.spinigerum. However, the number is less than 200 in most G.d%resi. On the other hand, the cervical papillae and excretory pore of G.hispidum were situated more anteriorly than those of above two species. In the present study, we compared the larvae from three districts, Temazcal, Sinaloa and Tepic, and found that those morphologies quite resemble that of G.spinigerum in Thailand. In adult specimens, the morphology is also very similar to that of G. spinigerum in Thailand (Koga et al. 1991). The most noticeable difference between G. spinigerum and the present Mexican species is the surface of the eggshell. The eggshells of G.spinigerum and other gnathostome species have many pits on the surface, and the shape of these pits is species-specific (Koga, 1996), while the eggshell of the Mexican species are not pitted. In North-Central, South America, 3 species of Gnathostoma have been recorded: G. procyonis (Chandler, 1942) from a raccoon in Texas, G.turgidum (Stossich, 1902, quoted in travassos, 1925) from an opossum, and G.americanum (Travassos, 1925) from Felis tigrina, the latter two in Brazil. The adults of these 3 species have multidigitate (4-5 teeth) spines of their anterior region immediately behind the head bulb, and have dense spines on the posteriorhalf of their body surfaces, except for G.turgidum. HQwever, G.turgidum has bipolar plugged eggs. In contrast, our specimens had very minute spines sparsely distributed over the posterior half of their bodies, and these spines were recognizable only by SEM examination. The eggs have unipolar plug. Therefore our new specimens are easily distinguishable from previously reported species in America. Consequently, a new species gives rise to occur the mexican gnathostomiasis. 3

References 1) Akahane, H., R. Lamothe-Argumedo, ]. M. Martinez-Cruz, D. Osorio-Sarabia, and L. Garcia-Priento. (1994): A morphological observation oí the advanced third-stage larvae oí Mexican Gnathostoma. ]apanese ]ournal oí Parasitology, 43, 18-22. 2) Almeyda-Artigas, R.]. (1991): Hallazgo de Gnathostoma binuc/etum n.sp. (NEMATODA: SPIRURIDA) en íelinos silvestres y el papel de peces dulceacuicolas y oligohalinos como vectores de la gnathostmiasis humana en la cuenca baja del rio Papaloapan, OaxacaVeracruz, Mexico. Anales del instituto de Ciencias del Mar y Limnologia, Universidad Nacional Autonoma de Mexico, 18, 137-155. 3) Ash, 1. R. (1962): Development oí Gnathostoma procyonis Chandler, 1942, in the first and second intermediate hosts. ]ournal oí Parasitology, 48, 298-305. 4) Camacho, S. P. D., Zazueta-Ramos, E. Ponce- Torrecillas, 1. Osuna-Ramires, R. CastroVelazquez, A. Elores- Gaxiola, ]. Baquera-Heredia, K. Willms, H. Akahane, K.Ogata, and Y. Nawa. (1998): Clinical Maniíestations and immunodiagnosis oí gnathostomiasis in Culiacan, Mexico. American ]ournal oí Tropical Medicine and Hygiene, 59,908-915. 5) Chandler, A. C. (1942): The helminths oí raccoons in east Texas. ]ournal oí Parasitology, 28, 225-268. 6) Koga, M. and Y. Ishii. (1987): Surface morphology oí the advanced third-stage larvae of Gnathostoma doloresi - An electron microscopic study-. ]apanese ]ournal oí Parasitology, 36, 231-235. 7) Koga, M., ].Ishibashi, Y.Ishii, and T.Nishimura. (1988): Scanning electron microscopic comparisons among the early and advanced third-stage larvae oí Gnathostoma hispidum and the Gnathostoma larvae obtained from loaches. ]apanese ]ournal oí Parasitology, 37, 220226. 8) Koga, M., H. Akahane and Y. Ishii. (1991): Surface ultrastructure oí adults and eggs oí Gnathostoma spinigerum (Nema toda : Gnathostomatidae). Transactions oí the American Microscopical Society, 110, 315-320. 9) Koga, M., H. Akahane, and Y. Ishii and S. Kojima. (1994): External morphology oí the advanced third-stage larvae oí Gnathostoma spinigerum: A scanning electron microscopy. ]apanese ]ournal oí Parasitology, 43, 23-29. 10) Koga, M. (1996): Comparative surface ultrastructure oí adults and eggs oí Gnathostoma obtained in ]apan. Southeast Asian ]ournal oí Tropical Medicine and Public Health, 27, 71-74. 11) Lamothe-Argumedo, R., R.1. Medina- Vences, S. Lopez- ]imenez, and 1. Garcia-Prieto. (1989): Hallazgo de la íorma iníectiva de Gnathostoma sp., en peces de Temazcal, Oaxaca, Mexico, Anales del Institutio de Biologia de la Universidad Nacional Autonoma de Mexico., Series-Zoologia, 60, 311-320. 12) Ogata, K., Y. Nawa, H. Akahane, S. P. Camacho, R. Lamothe-Argumedo and A. Cruz Reyes. (1998): Gnathostomiasis in Mexico. American ]ournal oí Tropical Medicine and Hygiene, 58, 316-318. 13) Pelaez, D. and R. Perez-Reyes. (1970): Gnathostomiasis humana en America. Revista Latino-Americana de Microbiologia, 12, 83-91. 14) Travassos, L. (1925): Contrbuicoes para o conhecimento da íauna helmintologica brasileira. 18 Sobre as especies brasileiras do genero Gnathostoma Owen, 1836. Sciencia Medica, 3, 508-517. 4

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Figure regends Figs.1-6 and 10-17. Scanning electron micrographs. Fig.1. Lateral view oí the head bulb oí Gnathostoma binucleatum from Temazcal. The arrowhead indicates the cervical papilla. Fig.2. An enlarged riew oí the hooklets. The base oí each hooklets has an oblongate shape. Sharp keratin hooks armed posteriorly. Fig.3. A mammiform oí the cervical papillae protruding írom the tegumento Fig.4. An oval shaped opening oí the excretory pore which opened ventrally. Fig.5. The terminal end oí a larva. The anal opening is clearly visible on the ventral surface of the larve, which has a crescent shape. Fig.6. A terminal extremity oí a larva. A phasmidial pore can be laterally seen. Figs.7-8. Cross sections oí the larval intestines. Fig. 7. A cross section oí the Temazcal larva. Multiple nuclei were evident in one cell. Fig. 8. A cross section oí the Sinaloan larva. Arrowheads indicate the cells bearing 5 nuclei each. Fig. 9. Parasitic nodule (arrowhead) in the stomach wall of a dog iníected with the Mexican larvae. Fig.10. Lateral view oí the head buld of adult Ganthostoma binucleatum. Fig.11. Short, stumpy tridentate spines lying immediating behind the head bulbo Fig. 12. Mammiform cervical papilla located between the eighteenth and twentieth transverse striations, with spines of trididentates on the body surface. Fig. 13. Dome-shaped excretory pore. Around this pore, all spines are unidentate with acute tips. Fig. 14. Smaller unidentate spines located at middle part oí the body. Fig.15. Very minute spines (arrowheads) are sparsely distributed around the posterior half of the body. Fig.16. Ventral surface of the tail of the maleo Dome-shaped caudal papillae and some small papillae without spines are evident. Fig.17. An egg from the uterus oí a gravid female of Gnathostoma binucleatum bearing an operculum on one end, showing smooth surface. 8