Prevalence and egg surface morphology of capillariid nematodes in red foxes (Vulpes vulpes) from Hokkaido, Japan

Abstract

Capillariid nematodes, such as Aonchotheca putorii, Eucoleus aerophilus, E. boehmi, and Pearsonema plica, parasitize canids. Reports about these nematodes parasitizing red foxes (Vulpes vulpes) in Japan are limited, and there are no prior records of E. boehmi. This study investigated 56 red foxes captured in Hokkaido and detected eggs of the four above species and Calodium hepaticum in rectal feces, with E. boehmi showing the highest prevalence (48%) as the first report in Japan. Low-vacuum scanning electron microscopy revealed distinct egg surface textures that support the current microscopical species identification keys. This study reveals that capillariids are prevalent in foxes in Hokkaido and the high prevalence of E. boehmi suggests a potential infection risk to domestic dogs.

The family Capillariidae (Nematoda: Enoplida) contains numerous parasitic species in all vertebrate classes. While they were historically grouped under the genus Capillaria, current taxonomy recognizes 20 or more genera within this family [10, 11, 19]. Canids are parasitized by four major capillariid species: Aonchotheca putorii (Rudolphi, 1819) López-Neyra, 1947 in the stomach, Eucoleus aerophilus (Creplin 1839) Dujardin, 1845 in the trachea and bronchi, Eucoleus boehmi (Supperer, 1953) Moravec, 1982 in the nasal cavity and paranasal sinuses, and Pearsonema plica (Rudolphi, 1819) Moravec, 1982, in the urinary bladder [4, 5]. In practice, eggs from Calodium hepaticum (Bancroft, 1893) Moravec, 1982, which parasitizes the livers of mainly rodents, are often detected in the feces of canids due to their predation of infected rodents [4, 22]. Capillariid eggs have distinctive eggshell surface structures, which distinguishes them from the similarly shaped (barrel- or lemon-shaped with bipolar plugs) but smooth-surfaced eggs of Trichuris spp. These characteristic eggshell surface morphology differences can be used as key diagnostic features to differentiate capillariid species [3,4,5,6, 19].

In Japan, A. putorii has been found in various carnivores including raccoon dogs, raccoons, cats, Japanese badgers, and Japanese martens, Japanese weasels, Siberian weasels, sables and American minks [16, 24, 27]. Eucoleus aerophilus has also been reported from martens, Tsushima leopard cats and Iriomote cats [20, 29, 30], and P. plica has been reported from martens [21]. Furthermore, regarding capillariid nematodes from canids in Japan, Tamau et al. (2025) recorded three new species from raccoon dogs, Aonchotheca suzukii in the stomach and small intestine, Echinocoleus yokoyamae in the stomach, and Pearsonema neoplica in the urinary bladder [24]. However, there are few records of capillariid nematodes from red foxes (Vulpes vulpes) in Japan, and no available reports of E. boehmi.

In Hokkaido, intestinal capillariids have been recorded as Capillaria sp. from red foxes [8]. Meanwhile, capillariid eggs have been frequently detected in fecal surveys of foxes, and these have been assumed to be Calodium hepaticum resulting from pesudoprasitism [13]. This assumption is supported by the fact that foxes prey on a large numbers of voles (e.g. average 6 per day) and other rodents [1] and C. hepaticum infection is frequently found in wild rodents [15, 28]. However, this assumption has not been verified, and non-intestinal capillariids have been rarely investigated in red foxes in this area. Therefore, this study aimed to comprehensively reveal the presence of capillariid species in red foxes in Hokkaido, Japan. In addition, the fine structure of the egg surfaces from the nematode specimens were observed using a low-vacuum scanning electron microscope (LV-SEM) to confirm the morphological keys based on egg surface structure.

A total of 56 red fox carcasses (22 adult females, 20 adult males, 14 juveniles), culled between 2003 and 2005 on Hokkaido Island, Japan, were used for the survey (Supplementary Table 1). The foxes were trapped or shot by local hunters as part of the local administration policy to mitigate crop damage in accordance with the Act on the Protection and Management of Wildlife, and the Optimization of Hunting. Of 56 foxes, 38 (9 adult females, 15 adult males, 14 juveniles) were collected at Otaru in the middle of Hokkaido. Remaining foxes were all adults; 4 females from the southern and 9 females, 5 males from the eastern area (Supplementary Fig. 1). The carcasses were frozen at −80°C for more than 2 weeks to inactivate Echinococcus multilocularis eggs, then preserved at −20°C until examination. Fecal samples were collected from the rectums or colons of the carcasses and 0.5 g of each fecal sample was examined using a sucrose centrifugal floatation technique (specific gravity=1.27) [14]. The capillariid eggs were detected by microscopic examination and their species were differentiated based on morphological characteristics identified in the literature, especially the egg surface (Supplementary Table 2) [4,5,6, 17, 23, 24].

The digestive tracts, tracheas, and lungs from all 56 carcasses and the urinary bladders from 31 carcasses were examined for the presence of capillariid nematodes. The digestive tract was cut longitudinally and the mucosa and tract contents were scraped and repeatedly sedimented with saline in a 200-mL glass bottle until the supernatant was clear. The sediments were then examined under a stereoscopic microscope. Scraped tracheal mucosa and diced lung into 1–2 cm pieces were sedimented together and examined in the same way. The sediment from scraped bladder mucosae was also examined in a similar manner. In addition, in 10 carcasses that had tested positive for E. boehmi eggs, the skull was examined for the nematode. After cutting the frontal skull, the nasal cavity and sinuses were observed and the mucus was scraped and washed with saline in a glass dish to find the nematode. The collected capillariid nematodes were fixed in 70% ethanol, clarified in lactophenol, and identified to the species level based on morphological features found in the literature [5, 19, 23, 24].

Eggs from the four capillariid species were extracted from the uteri of fixed female worms that had been removed from the red foxes. For morphological observation, a single female specimen of each species was used: E. boehmi, E. aerophylus, and Aonchotheca sp. were obtained from foxes in Otaru, and Pearsonema sp. was from a fox in Shibetya. In addition, C. hepaticum eggs were taken from the uterus of a fixed female specimen collected from the liver of a gray-sided vole (Clethrionomys rufocanus) in Otaru. The nematode eggs were observed under a conventional microscope and the surface structure of the eggs was observed using an LV-SEM (Hitachi Miniscope TM1000, Hitachi Ltd., Tokyo, Japan).

Statistical analysis was performed to evaluate the effects of age and sex on the prevalence from the fecal egg examinations and autopsies using Fisher’s exact test in GraphPad Prism 10 (GraphPad Software, Boston, MA, USA).

Capillariid eggs were detected in 33 out of the 56 fecal samples (59%) from the red foxes. These eggs were differentiated based on morphological characteristics as E. boehmi in 27 samples (48%), Aonchotheca sp. (A. putorii or A. suzukii) in 9 (16%), E. aerophilus in 4 (7%), and C. hepaticum in 2 (4%) (Table 1, Fig. 1a, 1b). No P. plica or P. neoplica eggs were detected. The eggs from two capillariid species were detected in 9 samples (16%), and no samples contained eggs from more than three species. To evaluate the effect of age on the prevalence, adults and juveniles collected from Otaru (the only area where all juveniles were obtained) were compared. Fisher’s exact test showed no significant difference in the positive rates between the two age groups (P>0.05, Supplementary Table 3). Similarly, to examine the effect of sex, the positive rates were compared between adult males and adult females across all study areas and no significant differences were observed between the sexes (P>0.05, Supplementary Table 4). Detailed examination results for individual foxes are listed in Supplementary Table 1.

Table 1. Detection of capillariid nematodes in red foxes in Hokkaido.

Species	Fecal egg examination	Autopsy examination
	Percent prevalence (No. positive /No. examined)	Site	Percent prevalence (No. positive /No. examined)	Autopsy positive /Fecal egg positive	Mean Intensity ± SE (range)
Eucoleus boehmi	48 (27/56)	Nasal cavity and sunus	-a	9/10	18 ± 8 (1–79)
Eucoleus aerophilus	7 (4/56)	Trachea and lung	13 (7/56)	3/4	3 ± 3 (1–9)
Aonchotheca sp.	16 (9/56)	Stomach	16 (9/56)	6/9	52 ± 25 (1–250)
Pearsonema sp.	0 (0/56)	Urinary bladder	10 (3/31)	-	8 ± 2 (5–12)
Calodium hepaticum	4 (2/56)	NEb	-	-	-

^a 10 fox samples positive for eggs of E. boehmi were examined. ^b Not examined (pseudoparasitism).

Fig. 1.

Capillariid eggs from red foxes. Representative images of five capillariid species eggs. (a) and (b): light micrographs of eggs recovered from feces, (a) focused on the median plane and (b) surface. (c) and (d): light micrographs of eggs obtained from the uterus of female worms, (c) focused on the median plane and (d) surface. (e) and (f) low-vacuum scanning electron micrographs of the surfaces of eggs from the uterus. Scale bar: 20 µm (a–e), 2 µm (f).

After autopsy examinations of all 56 foxes, Aonchotheca sp. was detected in the stomachs of 9 foxes (16%) and E. aerophilus was found in the trachea and lungs of 7 (13%) (Table 1). The worm counts ranged from 1 to 250 for Aonchotheca sp. and 1 to 9 for E. aerophilus. Pearsonema sp. (P. plica or P. neoplica) was detected in the urinary bladders of 3 (10%) of the 31 examined foxes and the worm counts ranged from 5 to 12. Eucoleus boehmi was found in the nasal cavities and sinuses (Supplementary Fig. 2) of 9 of the 10 foxes (90%) in which its eggs had been detected by fecal examination. The worm counts ranged from 1 to 79. Worms of two capillariid species were detected in three foxes, and those of three species were found in two foxes. As with the results of the fecal egg examinations, the positive rate of each capillariid species was not significantly different between age groups or sexes (Supplementary Tables 3 and 4).

The morphology of the eggs obtained from the female adults was consistent with that of the eggs detected in the feces (Fig. 1). Images obtained using LV-SEM clearly demonstrated the differences in egg surface morphology among the capillariid species (Fig. 1e, 1f). The E. boehmi eggs had a uniquely pitted surface structure, whereas the other species had various net-like surface patterns: thick and coarse in Pearsonema sp. (P. plica or P. neoplica), thin and coarse in Aonchotheca sp. (A. putorii or A. suzukii), relatively fine in E. aerophilus, and distinctly fine in C. hepaticum.

This study has revealed the distribution of capillariid nematodes in red foxes in Hokkaido, including the first record of E. boehmi in Japan. The positive rate of E. boehmi (48%) was the highest on the fecal test, while that of C. hepaticum was the lowest (4%). The results can disprove the assumption that frequently detected capillariid eggs from red foxes in Hokkaido were primarily C. hepaticum [13]. Eucoleus boehmi has been reported in wild canid species in Europe and North America, with prevalences ranging from 8% to 94% [9]. The results from this survey suggested a moderately high prevalence of E. boehmi in foxes in Hokkaido compared to other areas.

Regarding the prevalence of E. boehmi in red foxes, Hodžić et al. (2016) reported no significant differences in the prevalence associated with age or sex [12]. In contrast, Germitsch et al. (2020) reported no significant difference between juveniles and adults, whereas males were more frequently infected than females [9]. Although the sample size in the present study was relatively small, no significant differences were observed in the prevalence of capillariid nematodes, including E. boehmi, between age groups or sexes. This suggests that juvenile foxes have opportunities for infection similar to those of adults, and that differences in hormones or behavior between the sexes may not substantially affect the frequency of infection.

Symptomatic cases of E. boehmi infection in dogs showing chronic nasal disorders, and more rarely, intracranial migration have been sporadically reported in Europe and North America [4, 7, 9]. The nematode transmission route to dogs has not been demonstrated, but it is thought to occur through the ingestion of earthworms (suspected intermediate or paratenic hosts) or eggs containing infective third-stage larvae [6]. On Hokkaido, red fox habitats overlap with the areas where dogs walk and play, such as parks and the urban periphery [26]. The high prevalence of E. boehmi in foxes revealed by this study suggests a potential risk of infection to dogs and clinicians should be aware of this possibility when encountering cases of nasal disease. As well as E. boehmi, clinical lung distress in dogs and cats by E. aerophilus infection has been reported [4, 25], suggesting a similar risk of infection from foxes to dogs and cats. In clinics, capillariid eggs are not commonly differentiated to the species level, and many cases might be misdiagnosed with Trichuris spp. [9, 25]. In such cases, the animals will not receive appropriate treatments. However, as demonstrated in previous reports [4, 6, 25] and this study, the eggs of these two respiratory capillariids can be distinguished from those of other capillariids (different surface textures) and Trichuris spp. (smooth surface) under conventional microscopic examinations.

It has been reported that the surface structures of capillariid eggs vary among species and can be used to identify major capillariid species that parasitize carnivores [4,5,6, 25]. In this study, the fine surface structures of eggs obtained from the uterus of each species were observed using LV-SEM. The obtained images clearly demonstrated various netted and pitted surface patterns on the eggs, which agrees with the egg differentiation described in previous studies [4, 6]. The detailed LV-SEM images of egg surfaces provide valuable reference to support the interpretation of images obtained under conventional light microscope in clinical diagnosis and parasitological survey.

In the present study, the LV-SEM method enabled the direct observation of nematode eggs preserved in 70% ethanol. However, the LV-SEM image resolution is generally lower than that of standard SEM. Furthermore, the wet and soft specimens used in this study were susceptible to vacuum effects and some eggs were deformed during observation. These effects probably caused the imaging differences between LV-SEM and standard SEM. For example, larger surface pits on E. boehmi were observed in this study compared to those shown in previous reports that used standard SEM [12, 18]. In addition, as a limitation of this study, the freezing and storing of carcasses and rectal feces can affect the egg morphology of these species, such as the space between the embryo and the egg wall and the egg surface structure [2, 9]. Consequently, the morphological features of fresh, unfrozen eggs would differ from the images of eggs shown in Fig. 1.

In summary, this study investigated the prevalence of capillariid species in red foxes from Hokkaido, Japan and the moderately high prevalence of E. boehmi is a novel finding. Furthermore, the results showed that there were distinct surface structures on the eggs of these species, which are useful for diagnostic purposes.

Conflicts of Interest

The authors declare that they have no conflict of interest regarding the content of this manuscript.