Seasonality of the nematode Gnathostoma turgidum in Virginia opossums (Didelphis virginiana) in the wild has been reported; however, the mechanisms involved in deworming are unknown. We monitored the parasitologic and biologic changes in four Virginia opossums naturally infected with G. turgidum by coproparasitologic examination and abdominal ultrasonography. Eggs became detectable in the feces of opossums in May, peaked in July and August, and suddenly decreased in October. Adults of G. turgidum were expelled in the feces mainly in September. Ultrasonography of the liver showed slight damage during May. Lesions in the stomach appeared in April and persisted until September. The abnormalities of the liver and stomach were resolved in November. These data suggest that G. turgidum is likely expelled as a result of host immunologic mechanisms, although termination of a natural life span cannot be definitively excluded.
Gnathostoma turgidum Stossich, 1902, is a gastric nematode that occurs in several species of opossums, including the Virginia opossum (Didelphis virginiana) in several countries of the Americas (Johnson 1970). In Mexico, G. turgidum is present in several states, including Guerrero, Chiapas, and Oaxaca (Lamothe-Argumedo et al. 1998). Recently, Tecualilla, in the municipality of Escuinapa, Sinaloa State, Mexico, was shown to be an enzootic area of G. turgidum, with a prevalence of more than 50% in opossums (Díaz-Camacho et al. 2009). During a >1-yr longitudinal survey in this area, pronounced seasonal changes of infection were observed in opossums. These changes included synchronized maturation of larvae to young adults in the liver, with subsequent relocation to the stomach to become mature adults (Nawa et al. 2009). All opossums captured from October to December were uninfected with G. turgidum.
We investigated whether “spontaneous cure” of G. turgidum in D. virginiana is a component of the parasite-host interaction. Naturally infected opossums were captured and chronologic changes in G. turgidum infection were monitored by coproparasitologic examination and ultrasonography of the liver and stomach.
With permission from the Mexican Wildlife Animal authorities (Dirección General de Vida Silvestre, SEMARNAT, 02197/12), we captured four opossums in February 2011 using hunting nets and night lights near the small water reservoirs of Ojo de Agua and Tanque Via, in Tecualilla, Escuinapa, Sinaloa, Mexico (22°45′28″N, 105°40′25″W). The captured animals were marked (1T–4T), and their sex, body weight, and other morphometric data recorded. A male opossum captured in a nonenzootic area in the municipality of Navolato, Sinaloa, Mexico (24°45′55″N, 107°42′7″W) was used as a negative control (5T). In addition, one opossum killed by local hunters in the same enzootic area described above in March 2013 was packed in ice, transported to our laboratory, and examined within 48 hr of being killed. This opossum was used for confirmation of the ultrasonographic imaging and actual visceral lesions. After ultrasonographic imaging examination, the thoracic and abdominal viscera were examined visually for nematodes.
Live animals were transported to the laboratory and kept under natural temperature, humidity, and light conditions, with no access to external food sources. They were housed individually in 50×50-cm wire-mesh cages fitted with a catch pan for fecal collection and fed commercial dog food and water ad libitum. Catch pans were cleaned daily and fecal samples from each opossum were collected weekly. The presence of parasite eggs in stools was determined by flotation (Faust) and sedimentation (Ritchie) techniques (Beaver et al. 2003). For quantification of G. turgidum eggs, a McMaster chamber counting slide (Chalex Corp., Wallowa, Oregon, USA) was used following the manufacturer's instructions. Additionally, whole stools were visually examined daily before cleaning the catch pan to search for adult nematodes or fragments thereof.
Adult worms from opossum feces were measured using an electronic vernier caliper (Mitutoyo, Kawasaki, Kanagawa, Japan) and examined under a light microscope (Primo Star, Zeiss, Göttingen, Germany) to record the number of rows of hooklets on the cephalic bulb, proportion of the body covered with spines, and shape and height of the spines (Díaz-Camacho et al. 2009). The morphologic identification of adult G. turgidum was performed according to Bertoni-Ruiz et al. (2011).
The presence or absence of possible Gnathostoma-associated lesions was corroborated by abdominal ultrasonography (stomach and liver) using a veterinary digital diagnostic ultrasound system, DP-2200VET (Mindray, Mahwah, New Jersey, USA). The opossums were sedated using a 10 mg/kg intramuscular injection of tiletamine/zolazepam (Zoletil50, Virbac, Guadalajara, Jalisco, Mexico). Hyperechoic images suggestive of pathologic changes in the liver and gastric wall were recorded.
All captured animals weighed 1.1–1.3 kg at the beginning of the study. They exhibited unusual hair loss and a soft dirty coat. Naturally infected opossums generally gained approximately 800 g after spontaneous worm expulsion in December.
The results of weekly fecal egg examination are shown in Figure 1. Feces of all animals were negative for Gnathostoma spp. eggs from February to April, but other nematode eggs such as Turgida turgida and Strongyloides spp. were present. In the four naturally infected opossums, Gnathostoma eggs became detectable in May, rapidly rose to a peak around June, remained at a high level until September/October, and decreased sharply thereafter. No Gnathostoma sp. eggs were detected in feces of the negative control.
Abdominal ultrasonography revealed hepatic and gastric hyperechoic areas in all infected opossums (Fig. 2). In the liver, areas suggestive of damage were scattered in the parenchyma in various lobes from March (Fig. 2A) until August, but thereafter complete regeneration was observed in all infected animals. In the opossum killed by hunters in March 2013, visual examination revealed larvae and one immature adult G. turgidum in the liver (Fig. 2B). No nematodes were found in the stomach. Stomach ultrasonography of infected opossums revealed foreign bodies with apparent mobility, especially in July (Fig. 2C) and was consistent with the presence of eggs in the feces.
All naturally infected opossums gained weight during November and showed a marked improvement in the quality of hair after recovery of the hepatogastric lesions. The control opossum had bright, healthy, abundant hair, homogeneous liver parenchyma, and a healthy stomach throughout the study.
Twelve worms were expelled from the four Gnathostoma egg–positive opossums between the first week of May and mid-October, with the peak recovery during September (Table 1). Length of female worms ranged from 88 mm to 99 mm (mean 92.3 mm, n = 3); males were 44 mm to 79 mm (mean 60.5 mm, n = 6). The cephalic bulb in all adults was armed with uncinate conical base hooklets in eight transverse complete and two incomplete rows, and the posterior third of the body lacked cuticular spines.
Synchronized migration, beginning with the appearance of immature adults in the liver in early February, followed by their growth in the liver, relocation to the stomach, development to mature adults, and final disappearance in September has been documented (Nawa et al. 2009). In our study, captured opossums were infected with G. turgidum before they were caught in February, possibly by eating a frog or turtle infected with infective third-stage larvae, as previously suggested (Mosqueda Cabrera et al. 2009). Gnathostoma eggs appeared in the feces almost synchronously in early May. From June to August, persistent high counts of eggs per gram were observed. From August to October, live and dead adults were expelled. These results are in accordance with our previous observations of the seasonality of G. turgidum using a longitudinal survey of wild opossums in the same area (Nawa et al. 2009).
It appears that the seasonality of G. turgidum infection in opossums is a result of a host-parasite interaction. Similar seasonality occurs with Gnathostoma procyonis in raccoons, (Procyon lotor; Lockhart 2007). Although persistent infection in the definitive host was reported for Gnathostoma spinigerum (Miyazaki 1960), a slight seasonal fluctuation was observed in experimentally infected dogs (Maleewong et al. 1992). The sudden termination of G. turgidum infection in the opossums in our study is unlikely to reflect the lifespan of the parasite because the majority of expelled adults were still alive and the females were laying eggs. Thus, the host immune system may have achieved immune-mediated deworming. Many examples of expulsion in natural and experimental infections have been reported for intestinal nematodes (Else and Finkelman 1998). In particular, the importance of heparin-binding proteins (Maruyama et al. 2000) and proteoglycans produced by intestinal goblet cells and mast cells (Nawa et al. 1994) has been demonstrated. Our results show that after a period of egg production, the adult nematodes were expelled in the feces, and the hepatic and gastric lesions healed. Confirmatory experiments regarding the involvement of the immune system in the seasonality of G. turgidum in opossums are currently underway in our laboratory.
This work was supported by institutional funds (PROFAPI-UAS2009/176, 2011/139, and 2012/145). E.H.T.M. was supported by CONACYT scholarship 103950. We thank Mónica González-Lázaro for reviewing the use of English in the manuscript.