Improved molecular identification of Strongyloides myopotami in nutrias using fecal samples

Abstract

Strongyloides myopotami is an intestinal nematode parasite of nutrias. Identification of S. myopotami is conducted based on the morphological characteristics of adult worms or cultured larvae. To widely and effectively understand the infection in nutrias, it would be preferable to develop the molecular identification using a few grams of the feces. Here, we attempted to identify S. myopotami using DNA extracted from eggs obtained from fecal samples. Among previously reported primer pairs targeting the 18S rRNA gene of Strongyloides spp., most could not be successful. We newly designed primers that successfully amplified the partial sequences in S. myopotami, resulting in being sequenced. Our simple protocol can be useful in nationwide surveys for clarifying the risk of human infection.

The nutria (Myocastor coypus), which is also known as the coypu, is a semi-aquatic rodent indigenous to southern South America [9]. The species is generally thought to be herbivorous, feeding mainly on plants with the occasional ingestion of insects, mussels, and crustaceans [14]. During the early 20th century, the animals were highly regarded as a source of fur and meat, and were introduced to many countries outside of their native range, including North America, Europe, and Japan [4]. However, some nutrias subsequently escaped into the wild or were released into the wild due to the decline of the fur industry, and as a result of their high adaptability to a wide variety of environmental conditions, there are now established wild populations of nutrias in many areas of the world, except for in Australia and Antarctica [11].

Nutrias often devastate natural communities by feeding on plants, including endangered ones, devouring crops, and burrowing in fields, resulting in serious damage to agricultural draining systems [2]. Due to their harmful impacts on native plant biodiversity and natural ecosystems, nutrias are recognized as one of the worst invasive alien species worldwide, including in Japan, by the International Union for Conservation of Nature and the European Union [13, 16]. Additionally, nutrias are known carriers of various epizootic and/or zoonotic pathogens, including bacteria and parasites, and are thus implicated in the transmission of diseases to livestock and humans [2, 17, 26]. However, the distributions of pathogens among the nutrias in different countries have not been fully clarified.

Since the 1960s, the number of feral nutrias in Japan has continually increased, mainly in the central and western parts of Honshu Island (the main island of Japan), and it has been suggested that they are spreading into more areas [20, 21]. Many local governments have been actively trying to control the number of nutrias to reduce the agricultural damage they cause. As part of the extermination programs in four prefectures of Honshu Island (Aichi, Gifu, Okayama, and Hyogo Prefectures), the intestinal contents or feces of captured nutrias were examined, and Strongyloides myopotami (Secernentea: Strongyloididae) was frequently detected with a prevalence of 98% to 100% [1, 19, 22]. Previously, S. myopotami has been reported to be a zoonotic parasitic nematode that causes a severe pruriginous rash in humans [3]. Unlike Strongyloides stercoralis, which infects humans and a variety of mammals and sheds larvae in feces, S. myopotami eggs containing divided or non-divided egg cells can be found in the feces of infected nutrias [5, 23]. Although infections may occur via percutaneous or oral routes from contact with larvae-contaminated soil or water, the detailed lifecycle of S. myopotami remains unknown. In most previous reports, S. myopotami has been identified by light or electron microscopy based on the morphological features of adult worms or larvae after the cultivation of feces or intestinal contents, and these adult worms were collected from the intestine during autopsy. In only one report, S. myopotami was identified by molecular analysis with the DNAs derived from an adult (Kyoto) and a cultured larva (Hyogo), which were derived from nutrias captured in Japan [10]. Although these results suggest a high prevalence of infection by the parasites among the nutrias in Japan, further investigations have not yet been conducted.

To facilitate studies on the parasitic infections in nutrias, it would be preferable to examine the parasites and identify the species using only a few grams of the feces collected for conventional fecal examinations, as an alternative noninvasive manner without the need for euthanasia or larva cultivation. Thus, in the present study, we tried to develop an easier method for detecting S. myopotami infection by molecular analysis targeting DNA that is extracted directly from eggs in the feces of nutrias. First, by using fecal samples of nutrias, we tried to amplify the genes by PCRs with previously reported PCR primers which were used for molecular analyses of adults or larvae of Strongyloides spp. including S. myopotami [6, 8, 10, 12], then we newly designed primers and evaluated their utility for PCR amplifications and sequencing analyses.

Eleven fecal samples of nutrias (Samples A to K) were examined. The feces were collected from nutrias in Okayama Prefecture (Samples A to G) and Osaka Prefecture (Samples H to K) from December 2020 to June 2021, which are located in the western part of Honshu Island, Japan. Most of the nutrias were captured as part of nutria extermination programs or pest control programs of two local governments of Okayama and Osaka Prefectures. Only Sample B was collected as freshly dropped feces, and the other samples (Samples A, and C to K) were collected as intestinal content samples mainly from the colon or rectum of nutrias. All samples were kept at 4°C for 3 to 4 weeks at most until examined.

The collected samples were examined by the modified sugar flotation method as previously reported [18]. Briefly, 1 g of sample was diluted in 10 mL of water, then filtered through gauze to remove large debris. The filtrated solution was centrifuged at 1,700 × g for 5 min, and the supernatant was discarded. A sugar solution with a specific gravity of 1.2 was added to the sediment. After centrifugation at 1,700 × g for 10 min, the surface was transferred to a glass slide using a nichrome wire loop, and covered with a slide cover glass. The entire smear was examined by light microscopy under 200× or 400× magnification.

After the parasitic examination, the eggs of positive samples (Samples A to K) (Fig. 1) were purified by the sugar flotation method using the remaining feces [7]. Briefly, 5–10 g of the fecal samples were diluted in water, and filtered through a steel mesh. After centrifugation at 1,700 × g for 5 min, the sugar solution was added to the sediment, and overlaid with water. After centrifugation at 1,700 × g for 10 min, the eggs that floated to the surface of the sugar solution were recovered using a Pasteur pipette, and washed with water one or two times. Finally, the purified eggs were resuspended in 1 to 2 mL of phosphate-buffered saline (PBS) and stored at 4°C. The population of the purified eggs was determined by counting number of the eggs using 5–20 μL suspensions under a microscope.

Fig. 1.

A nematode egg that was detected in the feces of a nutria. The scale bar indicates 10 μm.

Glass beads (0.5 to 0.7 mm; As One, Osaka, Japan) were added to 200 μL of the nematode egg solution at a ratio of 1:1 of the volume. The numbers of eggs in 200 μL of Samples A to K were 1,150, 25, 850, 100, 10, 100, 600, 37, 300, 25, and 12.5, respectively. The samples were subjected to freeze-thaw cycling five times. At every step before freezing after thawing, the eggs were broken by vortexing for approximately 10 sec at room temperature. Subsequently, the supernatants were collected, and the DNA was extracted using a QIAamp DNA Mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions.

In a previous study, species identification of adult or larval nematodes by molecular methods has been performed by PCR amplification of a partial sequence of the 18S ribosomal RNA (rRNA) gene covering four hyper-variable regions (HVR-I to -IV), and subsequent sequencing of the amplicons [10]. Thus far, nine specific primer pairs containing one to four regions among HVR-I to -IV (Table 1) have been reported for this purpose [6, 8, 10, 12]. In the present study, we first evaluated the previously reported primers using Samples A, B, and C, as briefly described below. PCR reactions were performed with Ex Taq DNA Polymerase (Takara Bio, Kusatsu, Japan) in a total volume of 25 μL containing 2 μL of template DNA and the following cycling conditions: an initial denaturation at 94°C for 1 min, followed by 10 cycles of 94°C for 1 min, 40°C for 1 min, and 72°C for 2 min, 30 cycles of 94°C for 1 min, 45°C for 2 min, and 72°C for 2 min, and post-amplification extension at 72°C for 7 min. The PCR products were electrophoresed on a 1.5% agarose gel, and visualized by ethidium bromide staining. The PCR products were then purified using the Viogene Gel/PCR™ DNA Isolation System (Viogene, Sunnyvale, CA, USA), and sequenced in both directions using amplifying primers. The obtained sequences were analyzed using the Basic Local Alignment Search Tool (BLAST) of the National Center for Biotechnology Information.

Table 1. Primers used for the identification of Strongyloides myopotami in the present study.

For the second part of the molecular identification study, we designed three new primer sets (StMyo F1-R1, StMyo F2-R2, and StMyo F3-R3; Table 1) that can bind to highly conserved sequences flanking the HVRs of the 18S rRNA gene in S. myopotami (AB453313) and other Strongyloides spp. The primers were expected to amplify sequences that include at least two regions among HVR-I to -III. PCR reactions using Samples A to K were conducted as described above with the exception that 0.5 μL of template DNA was used, and the cycling conditions were as follows: an initial denaturation at 94°C for 5 min, followed by 35 cycles of 94°C for 30 sec, 60°C for 30 sec, and 72°C for 1 min, and post-amplification extension at 72°C for 7 min. The PCR products were sequenced and analyzed as described above.

To evaluate whether PCR with the newly designed primers could amplify DNA from a small number of eggs, 1 or 10 eggs were picked from the purified egg solutions of Samples D and E using a Cell Picker^TM (Shimadzu, Kyoto, Japan). Five samples with a single egg, and three samples with 10 eggs were collected in 200 μL of PBS, then the DNA was extracted, and PCR was performed as described above.

Civil servants employed by the prefectural governments of Okayama (Okayama Prefectural Bizen Public Healthcare Center) and Osaka (Research Institute of Environment, Agriculture and Fisheries) Prefectures conducted the examinations of the animals. All examinations in this study were approved by the prefectural governments, and were conducted as a part of governmental affairs. No human samples were involved in this study.

By fecal examinations, nematode eggs as shown in Fig. 1 were found in all of 11 nutria fecal samples, and oocysts of Eimeria spp. were also detected in 5 samples. So far, the eggs in feces of infected nutrias were detected as those of different cleaved stages [5, 23]. In the present study, we found the eggs containing larvae (sometimes embryonated). Although the reasons for differences of the egg stages remains unknown, the temperature might not be controlled after the nutrias were captured and the feces were collected. To identify the species of the eggs, PCR was performed using previously reported primer sets, i.e., nine primer sets, including five forward primers and two reverse primers (Table 1). Six of the nine primer sets resulted in amplicons of the expected sizes in all of the tested samples (Samples A to C). The sequences from only four, zero, and three primer sets in Samples A, B, and C, respectively, were identical to those of S. myopotami (AB453313; Table 2). The sequences of the other expected products could not be determined due to mixed sequence signals or were found to be derived from other microorganisms, including bacteria, such as Pseudomonas spp. and Flavobacterium spp. Additionally, many nonspecific bands (bands of unexpected sizes) were confirmed in all samples by gel electrophoresis (Table 2).

Table 2. Results of PCR amplification and sequencing of 18S rRNA genes in nutria feces using previously reported primers.

Primers			Samples
Forward	Reverse	Expected size (bp)	A			B			C
			Expected*1 amplicon	Sequences*2	Nonspecific*3 bands	Expected*1 amplicon	Sequences*2	Nonspecific*3 bands	Expected*1 amplicon	Sequences*2	Nonspecific*3 bands
Nem18SF	18SPCR	1,500	-	/	+++	-	/	++	+	-	+++
18SP2F	18SPCR	950	+	-	+++	+	-	+++	+	-	+++
SSU24HF	18SPCR	750	+	Sm	+++	-	/	+++	+	Sm	+++
18SP4F	18SPCR	710	+	Sm	+++	+	-	+++	+	Sm	+++
SSU23F	18SPCR	320	+	-	+++	+	-	++	+	-	+++
Nem18SF	18SP8R	1,000	+	Sm	++	-	/	+	+	Sm	++
18SP2F	18SP8R	550	+	Sm	++	+	-	++	+	N.D.*4	++
SSU24HF	18SP8R	250	+	-	-	+	-	++	+	-	+
18SP4F	18SP8R	210	+	-	-	+	-	-	+	-	+

*¹ Expected amplicon. + and -: a band of the expected size was or was not present, respectively. *² Sequences. /: not done, Sm: identical to those of S. myopotami; -: not determined due to multiple sequence signals or high homology with other microorganisms. *³ Nonspecific bands. -: 0 bands; +: 1 band; ++: 2 to 5 bands; +++: more than 6 bands. *⁴ N.D.: not determined due to the presence of 2 to 5 nonspecific bands around the expected amplicon.

To improve the method of identification by PCR using the DNA of eggs derived from fecal samples, we designed three new primer sets that included at least two regions among HVR-I to -III. With the new primers, amplicons of the expected sizes were successfully detected in all 11 tested samples (Samples A to K) by PCR, although one unexpected band was found in Sample B with only one primer set (Table 3). All the sequences were completely identical to those of S. myopotami (AB453313).

Table 3. Results of PCR amplification and sequencing of 18S rRNA genes in nutria feces using newly designed primers.

Primers			Samples
Forward	Reverse	Expected size (bp)	A to K
			Expected*1 amplicon	Sequences*2	Nonspecific*3 bands
StMyo F1	StMyo R1	658	+	Sm	-
StMyo F2	StMyo R2	515	+	Sm	(−)*4
StMyo F3	StMyo R3	526	+	Sm	-

*¹ Expected amplicon. +: band of the expected size was present. *² Sequences. Sm: identical to those of S. myopotami. *³ Nonspecific bands. -: 0 bands. *⁴ One nonspecific band was found only in Sample B.

To evaluate the detection limit of the PCR using the newly designed primers, the DNA extracted from 1 or 10 eggs was examined. With all three primer sets, bands of the expected sizes were seen in four of the five samples with a single egg, and in all three of the samples with 10 eggs. No nonspecific bands were found by gel electrophoresis. The sequences of these products were identical to those of S. myopotami (AB453313).

The nutria population has been growing as they spread in Japan, and they inhabit areas close to human living areas as synanthropes. So far, S. myopotami, a zoonotic nematode, has frequently been detected in nutrias in Japan [19, 22]. We attempted to develop a method for the identification of S. myopotami infection using DNA extracted from eggs in feces obtained from conventional fecal examinations. With PCR using the previously reported primers, we were unable to obtain amplicons of the expected sizes in all of the samples, and a large number of nonspecific bands were seen. Additionally, by sequencing analysis of the obtained expected amplicons, the sequences could not be determined due to mixed sequence signals or implicate the amplifications derived from DNAs of other microorganisms. In previous reports, the sensitivity of these primer sets was not described [6, 8, 10, 12] and the clear reason for our results remains unknown, and however, in the previous study, adults and larvae were washed with PBS before DNA extraction, which may have reduced the number of other microorganisms present, including bacteria. In the present study, the DNA of other microorganisms may have remained after DNA extraction, because the eggs were purified by the sucrose flotation method. In addition, it is possible that eggs possess a smaller amount of DNA than adults and larvae due to their smaller size (female adult S. myopotami are 3.1 to 5.2 mm × 30 to 42 μm in size; filariform larvae are 645 to 800 μm × 13 to 15 μm in size; and eggs are 51 to 66 μm × 21 to 27 μm in size) [5, 15]. Furthermore, it has been reported that PCR inhibitors, such as complex polysaccharides, bile, salts, lipids, and urate, may be present in fecal samples [24], which may have affected the PCR results.

We designed three new primer pairs that contained at least two HVRs, and they successfully amplified a partial sequence of the 18S rRNA gene of S. myopotami. Moreover, PCR with these primers could produce amplicons from the DNA extracted from a very small number eggs, i.e., 1 to 10 eggs. Our results indicated that PCR using the newly designed primers is suitable for the detection of DNA derived from eggs of S. myopotami in feces. In the present study, we could not evaluate the amplification of other Strongyloides spp., and however, these primers might be applicable to them, since they were designed for highly conserved regions of the 18S rRNA gene of Strongyloides spp. Specifically, among eleven species of Strongyloides spp. which sequence data were available for alignments [10], sequences of StMyo F1, R2, and F3 were completely conserved, and those of StMyo R1, F2, and R3 were 3–4 bp differences in 3 species, 1 bp differences in 6 species, and 1 bp differences in 2 species. Additionally, we increased the annealing temperature by 15°C to 20°C in comparison to the PCR conditions for the previously reported primers. It has been reported that an increased annealing temperature can prevent the amplification of nonspecific bands [25]. These factors may have contributed to the successful amplification of the DNA extracted from feces.

In conclusion, we successfully developed an easier method for the identification of S. myopotami infection in nutrias. Nutrias are invasive alien species of global concern. The prevalence of S. myopotami has been investigated only in limited areas. In addition, S. myopotami has been reported in ctenomyid rodents [23]. Thus, further studies are needed to clarify the pathogenicity and transmission risk of S. myopotami in not only nutrias, but also humans and other animals.

CONFLICT OF INTEREST

The authors do not have any conflicts of interest to declare.