Possible zoonotic implications of the discovery of the advanced third stage larva of Gnathostoma turgidum (Spirurida: Gnathostomatidae) in a Mexican fish species

Human gnathostomiasis is a zoonotic disease with cosmopolitan distribution (Díaz-Camacho et al., 2020). Among the 13 valid Gnathostoma (Spirurida: Gnathostomatidae) species, advanced third-stage larvae (AdvL₃) of at least five species have been identified as etiological agents in this accidental human parasitosis: Gnathostoma spinigerum, G. doloresi, G. hispidum, G. nipponicum and G. binucleatum (Liu et al., 2020). According to these authors, humans can be infected through three routes: oral, trans-placental and through skin wounds. However, the most common via of human infection is through the accidental ingestion of AdvL₃ during the consumption of raw or undercooked fish meat.

This disease has been widely reported in the Mexican states of Veracruz, Tabasco, Sinaloa, Nayarit, Colima, Guerrero and Oaxaca, where local consumption of fish in the form of ceviche is very common (Ogata et al., 1998; Martínez-Salazar & León-Règagnon, 2005). Increased travel around the world, trade in fish from endemic areas, and its consumption in exotic dishes with raw or undercooked meat may promote gnathostomiasis in the Western Hemisphere (Bapat et al., 2022). In particular, three species of the genus Gnathostoma are distributed in Mexico: G. turgidum, G. binucleatum and G. lamothei (Díaz-Camacho et al., 2020). The AdvL₃ of two of these species infect the musculature of the second intermediate hosts in their life cycle, commonly fish (Herman & Chiodini, 2009; Nawa et al., 2015; Liu et al., 2020). In the case of G. turgidum, the first report of its larvae were made in the musculature of frogs (Mosqueda-Cabrera et al., 2009), and more recently in wild swamp eels (Monopterus albus) from Tampa, Florida (Cole et al., 2014). Among these species, until now only G. binucleatum has been shown to be the etiological agent of human infection (Díaz-Camacho et al., 2020). However, G. lamothei larvae have also been found in fish musculature (Hernández-Gómez et al., 2010), but to date there are no records that implicate this species as the causative agent of human infection.

The objective of this research is to document the first finding of the advanced third stage larva of G. turgidum in fish from Mexico and to discuss its possible role in human gnathostomiasis.

Bigmouth sleeper (Gobiomorus dormitor: Actinopterygii: Eleotridae) were obtained from fishmongers in Tlacotalpan, Veracruz at the beginning of the rainy season (August, 2022). The musculature of each host was examined by making thin fillets, compressed between two thick glass plates against the light, in order to locate nematode larvae. After the revision, the musculature of each fish was subjected to a digestion process in artificial gastric juice (6 g of pepsin, 7 ml. HCl in 1 liter of water) with a volume ratio of 3:1, at room temperature. The beakers used were covered with aluminum foil for 3 to 4 hours under constant agitation. Subsequently, the macerated tissue was removed and the rest was decanted by successive washings with 0.6 % saline solution; after each washing, the sediment was recovered until a clear solution was obtained that facilitated its observation under a stereoscope. The recovered larva was fixed in hot 70 % ethyl alcohol and kept in cold 70 % alcohol until study. For its identification (made on the basis of Mosqueda-Cabrera et al., 2009), the larva was cleared with Amman′s lactophenol; measurements (presented in micrometers) and images were obtained with a digital camera mounted on a compound microscope. The specimen was deposited in the Colección Nacional de Helmintos [CNHE], Instituto de Biología de la Universidad Nacional Autónoma de México, Mexico City, with the catalogue number: CNHE (11733).

The research related to animal use has been complied with all the relevant institutional policies for the care and use of animals in Mexico (DOF, 2001).

During examination against the light of the entire musculature of 17 G. dormitor [170 – 225 mm (206.82 ± 17.2 standard length)], a Gnathostoma larva went unnoticed and was only detected by pepsin digestion. This larva measures 1,544 by 142.3 long and wide, respectively. Cephalic bulb 61.3 length by 107.9 width covered by four rows of transverse spines with a single point. Each row has 34, 36, 40 and 45, from the first to the fourth row, respectively. The spines of the fourth row are on average smaller (5.9 μm) than those of the first row (7.4 μm). Additionally, the bulb presented some scales without hooks after the fourth row (Fig. 1).Esophagus 624.8 long by 92.21 wide; occupies 40.4 % of the total length of the body. Four cervical sacs project from the base of the cephalic bulb (329.4, 344.4, 359.2, and 393.9 long), spanning 57.1 % of the length of the esophagus. The entire body is covered with one-point spines, arranged in 171 transverse rows. Two cervical papillae located laterally between 11th and 12th spine rows on the body; excretory pore in row 21. Genital primordium situated at 55.7 %. Two caudal papillae lateral to the body, both posterior to the genital primordium; the right at 56.5 %; the left to 70.4 %. Anus to 1.75 % of posterior end of body.

Fig. 1.

Additionally, the larval stages of the following species were found: Digenea: Clinostomum marginatum (Clinostomidae) and Diplostomum sp. (Diplostomidae), Nematoda: Contracaecum sp. (Anisakidae), Spiroxys sp., (Gnathostomatidae) and Serpinema trispinosum (Camallanidae), and Pentastomida: Sebekia sp. (Sebekidae).

The morphometric study of the AdvL₃ collected from G. dormitor in the Papaloapan River, Veracruz, allowed its identification as G. turgidum according to the description presented by Mosqueda-Cabrera et al. (2009); this larva differs from those of G. binucleatum (Almeyda-Artigas, 1991; García-Márquez et al., 2009) and G. lamothei (Gaspar-Navarro et al., 2013), by the following combination of characters: 1) smaller body size, 1,544 (1,670) vs. (4,300) and (4,488), respectively]; position of the excretory pore in the row, 21 [(19.7) vs. (30.0) and (23.1)]; 3) less number of hooks per row on the cephalic bulb, 31.8, 35.0, 37.4, 41.4 vs. 38.7, 42.4, 44.7, 48.2 in G. binucleatum and 39.3, 43.3, 44.2, 47.3 in G. lamothei; and finally, 4) different dimensions of the spines in the rows of the cephalic bulb, being smaller in the fourth row of G. turgidum with respect to the 2 mentioned species, whose spines are of homogeneous size throughout the entire bulb.

The discovery of the larva of G. turgidum in this species of eleotrid represents the first for the nematode in a wild fish in Mexico and the second at global level (see Cole et al., 2014). In addition, Mosqueda-Cabrera et al. (2009) experimentally demonstrated that 2 other fish species (Poecilia gracilis and Oreochromis niloticus) are suitable hosts for G. turgidum. This is particularly relevant for the etiology of the gnathostomiasis, since several authors have suggested the possibility that any species of this genus present in Mexico could infect humans (Almeyda-Artigas et al., 2000; Hernández-Gómez et al., 2010; Díaz-Camacho et al., 2020), which seems to be supported by the molecular phylogeny carried out by Bertoni-Ruiz (2006). Based on the 28S gene of 7 of the 13 species that composed the genus, this author found that the species that have been recorded as parasites of man: G. spinigerum, G. hispidum, G. doloresi, G. nipponicum and G. binucleatum (Miyazaki, 1991; Almeyda-Artigas, 1991; León-Règagnon et al., 2002) do not group in the same clade. According to Bertoni-Ruiz (2006), these results could suggest that other species of the genus are potentially infectious for humans, provided they involve an intermediate or paratenic host in their food chain. However, this conclusion was obtained with only 50 % of the species represented, so it is necessary to deepen the phylogenetic analysis of the group.

The bigmouth sleeper has been also found be parasitized by G. lamothei and G. binucleatum larvae in Mexico (Hernández-Gómez et al., 2010). The above, added to our finding of AdvL₃ of G. turgidum in this species of fish (whose commercial value in its area of distribution establishes it as a host with a high risk of transmission), makes it necessary to highlight the importance of the methods of collection and identification of larvae of Gnathostoma, both in their natural hosts and in man.

In general, two methods have been used for the collection of Gnathostoma AdvL₃ in Mexico. The most widely used method is the examination of the musculature of fish by compressing it between two glass plates and its observation against the light using a 100-watt light source (Álvarez-Guerrero & Alba-Hurtado, 2007), or under a stereoscopic microscope (Hernández-Gómez et al., 2008; Hernández-Gómez et al., 2010). The second method (artificial digestion with gastric juice prepared with commercial pepsin and hydrochloric acid) has been implemented only in some cases (Lamothe-Argumedo & Osorio-Sarabia, 1998). For finding of larvae with the first method, the size of the larva is determinant; the observation with the naked eye of a well-defined red or orange point (appearance of the coiled or encysted larva) in the muscle tissue allows its location. With artificial digestion, the larvae are recovered from the bottom of the solution, observed under a stereoscope. In the particular case of the 3 species distributed in Mexico, the body size of the larvae (in millimeters) is different: 2.6 – 5.9 (4.3) for G. binucleatum (Almeyda-Artigas, 1991), 3.6 – 5.1 (4.5) for G. lamothei (Gaspar-Navarro et al., 2013) and 1.5 – 2.0 (1.7) for G. turgidum (Mosqueda-Cabrera et al., 2009). Therefore, the probability that the larvae of the first two species will be detected using the backlight method is higher, while those of G. turgidum could go undetected.

Based on the findings of our study, we consider pertinent to continue the search for G. turgidum AdvL₃ in fish associated with water bodies where there is a record of adult forms, applying the artificial digestion method, as well as corroborating their specific identification using molecular markers. Additionally, it is highly recommended that the larvae extracted from human biopsies be identified at a specific level in order to know precisely the role of the three species of Gnathostoma in human infections in Mexico.