MORPHOLOGICAL AND MOLECULAR ...

TAP CHI SINH HOC 2017, 39(3): 270-275 Morphological and molecular characterisation of Strongyloides ransomi DOI: 10.15625/0866-7160/v39n3.9829

MORPHOLOGICAL AND MOLECULAR CHARACTERISATION OF Strongyloides ransomi (Nematoda: Strongyloididae) COLLECTED FROM DOMESTIC PIGS IN BAC GIANG PROVINCE, VIETNAM Nguyen Thi Huong Giang1, Tran Duc Hoan1, Nguyen Thi Thu Huyen1, Nguyen Thi Kim Lan2, Pham Ngoc Doanh3* 1

Bac Giang Agriculture and Forestry University 2 Thai Nguyen University 3 Institute of Ecology and Biological Resources, Vietnam Academy of Sciences and Technology ABSTRACT: The genus Strongyloides is a group of parasitic nematodes of vertebrates comprised of over 50 species. A large numbers of nucleotide sequences of Strongyloides species have been deposited in GenBank. The vast majority of them are of Strongyloides from human beings, nonhuman primates, rats and cattle, but there are few molecular data of Strongyloides species isolated from pigs. The aim of this study is to identify Strongyloides species collected from pigs from Bac Giang Province, Vietnam, and analyze their molecular phylogenetic relationship within the genus Strongyloides based on 18S rDNA sequences. The morphological and molecular analyses revealed that the Strongyloides samples collected from pigs from Bac Giang Province, Vietnam, were in fact S. ransomi. The 18S rDNA sequences of S. ransomi from Vietnam were completely identical with that from Cambodia, but slightly different (0.3%) from that of Japan. In comparison to other Strongyloides species, S. ransomi was genetically close to S. venezuelensis. Keywords: Strongyloides ransomi, morphology, molecular, Vietnam. Citation: Nguyen Thi Giang, Tran Duc Hoan, Nguyen Thi Thu Huyen, Nguyen Thi Kim Lan, Pham Ngoc Doanh, 2017. Morphological and molecular characterisation of Strongyloides ransomi (Nematoda: Strongyloididae) collected from domestic pigs in Bac Giang province, Vietnam. Tap chi Sinh hoc, 39(3): 270275. DOI: 10.15625/0866-7160/v39n3.10796. *Corresponding author: [email protected] Received 18 May 2017, accepted 20 August 2017 INTRODUCTION

The nematode genus Strongyloides is comprised of over 50 species, all of which are parasitic in vertebrates (Viney & Lok, 2015). The vast majority of them are the intestinal parasites of domestic mammals including livestock, cats, dogs and pigs. Strongyloides species have deleterious effects on performance of the hosts, causing marked reductions in growth rate, feed efficiency, utilization and retention of nutrients, sometimes leading to the death of host animals (Stewart et al., 1968; Hale & Marti, 1984). A large number (about 8,200 so far) of nucleotide sequences of Strongyloides species have been deposited in GenBank with the extreme predominance of S. stercoralis, S. fuelleborni, S. ratti, S. venezulensis and S. papillosus from human beings, non-human 270

primates, rats and cattle. However, there are few molecular data of Strongyloides species isolated from pigs. The aim of this study is to identify the Strongyloides species collected from pigs from Bac Giang Province, Vietnam, and to analyse its molecular phylogenetic relationship within the genus Strongyloides based on the hypervariable region (HVR) I of the 18S rDNA sequence. MATERIALS AND METHODS

Collection of samples Strongyloides adult worms were collected from the small intestine of domestic pigs from 5 districts (Viet Yen, Yen Dung, Hiep Hoa, Son Dong and Lang Giang) of Bac Giang Province, Vietnam. The worms were washed repeatedly with physiological saline. Some of them were

Nguyen Thi Giang et al.

fixed and stored in 100% ethanol at -20°C for molecular analyses. The rest of the worms were fixed in 10% formalin for morphological examination. Parasite identification, measurements and photography Strongyloides adult worms collected from pigs were first identified based on their morphology referring to the previous descriptions (Sandground, 1925; Schwartz & Alicata, 1930). Morphometric data of the worms were obtained using a calibrated eyepiece scale fitted on a light microscope. The photographs of the worms were taken using a Axiocam Erc 5s digital camera attached to a Axio Lab A1 microscope (Carl Zeiss, Oberkochen, Germany) and ZEN lite software (Carl Zeiss). DNA sequence analyses One each of Strongyloides adult worm samples (VN1-VN5) collected from 5 different pigs from 5 different districts (one pig/district) were used for DNA analyses. The worms fixed in 100% ethanol were washed with distilled water and transferred individually to Eppendorf tubes. The total DNA of individual worms were extracted using QIAamp DNA stool MiniKit (Qiagen, Hilden, Germany). The hypervariable region (HVR) I (about 863 bp) of the 18S rDNA (Small Subunit, SSU) sequence was ampliﬁed by PCR using the primer pair, SSU18A 5'AAAGATTAAGCCATGCATG-3' as a forward primer and SSU26R 5'-CATTCTTGGCAAAT GCTTTCG-3' as a reverse primer (Doris et al., 2002; Jaleta et al., 2017). The conditions used for PCR were: initial denaturation at 94°C for 1 min followed by 30 cycles of 94°C for 1 min, 52°C for 90 sec, 72°C for 1 min, and a postamplification extension for 7 min at 72°C. The PCR products were electrophoresed in a 1.0% agarose gel and visualized by ethidium bromide staining. The positive PCR products were sent to the Macrogen Co., (Seoul, Korea) for sequencing. Alignment and phylogenetic analyses Five nucleotide sequences of 18S rDNA of the samples were determined. These sequences

were deposited in the GenBank/EMBL/DDBJ nucleotide sequence database with the accession No. LC324899 - LC324893. In addition, eighteen sequences of several Strongyloides species were obtained from the GenBank nucleotide database, and the 18S rDNA sequence of Parastrongyloides trichosuri was used as an outgroup. The sequence data were set up for reconstructing the phylogenetic tree using MEGA 6 (Tamura et al. 2013). They were aligned with Clustal-W with default options. The phylogenetic tree was constructed using the Maximum Likelihood (ML) method. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.0500)). The statistical confidence of branching patterns was evaluated by the bootstrap test. RESULTS AND DISSCUSION

Morphology of Strongyloides from pigs Parasitic females (fig. 1). Body long, filariform with 4.5-5.0 mm in length and 52.560.0 µm in width (fig. 1a). The esophagus is 940-1,100 µm in length (fig. 1b). From the base of the esophagus, the body narrows gradually toward the anterior end, the width of the head becomes narrower with a diameter of 22.5-25.0 µm (fig. 1b). From the region of the posterior ovarian loops the body narrows considerably and gradually reduce the size towards the anus to make the conical tail with the pointed tip (fig. 1c). The vulva is a transverse slit, with salient lips, situated at a distance of 2.6-3.1 mm from the anterior end (fig. 1d). The anus is located at a distance of 55-75 µm from the posterior end (fig. 1e). The ovary is twisted in loops anteriorly and is less constantly looped posteriorly. The eggs are ellipsoidal with thin shell, with 44-48 µm in length and 24-28 µm in width. Molecular analyses of 18S rDNA sequences The 18S rDNA sequences obtained from 5 Strongyloides samples were 812 bp in length and were completely identical with each other. The analyses revealed that Strongyloides samples from pigs in Bac Giang Province, Vietnam, were completely identical with the 271

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sequence (KU724126) of S. ransomi from Cambodia, but slightly (0.3%) different from the sequence (AB453327) from Japan. In the phylogenetic tree (fig. 2), all S. ransomi sequences are grouped together to make a common branch close to S. venezuelensis with a slight difference of 0.3%. One sequence (AB453323) of S. papillosus is completely identical with S. venezuelensis sequences, and is

also placed in this group, whereas, two other identical sequences (AB923886 and EF066361) of S. papillosus are considerably different (1.1%) from the sequence AB453323 and close to S. fuellerborni group. The 18S rDNA sequences of S. stercoralis and S. procyonis show high similarity and are grouped in one clade.

Figure 1. Morphological features of Strongyloides ransomi adult females collected from pigs in Bac Giang Province, Vietnam a.Whole body; b. Anterior end; c. Posterior end; d. Vulva (arrow); e. Anus (arrow).

The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. New sequences obtained in this study are represented with the sample codes in bold and others from DNA database are indicated with the Accession No., species name, and 2 letter country code (KH= Cambodia, MM= Myanmar, JP= Japan, DE= Germany, GA= Gabbon, UK= England, US= USA, VN= Vietnam). DISCUSSION In the genus Strongyloides, 18S ribosomal DNA sequence is rather variable

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among species and is suitable for species identification (Dorris et al., 2002; Hasegawa et al., 2009), except for high homology between S. stercoralis and S. procyonis which can be differentiated by ITS1 and 28S sequences (Sato et al., 2006). The 18S ribosomal DNA sequence includes four hyper-variable regions (HVR-I to -IV). HVR-IV shows species-specific arrangements, except that S. ransomi from pigs and S. venezuelensis from rats have the same nucleotide sequence in HVR-IV but they are distinguished by the difference in HVR-I and – III (Hasegawa et al., 2009). In this study, based on the analysis of morphology and the HVR-I sequences, we identified Strongyloides from pigs from Bac Giang Province, Vietnam, as S.


ransomi. Before this study, only three 18S rDNA sequences of S. ransomi (2 from Cambodia and 1 from Japan) are available in GenBank. One sequence (KU724126) from Cambodia is short and was excluded from this study. The sequences of S. ransomi from Bac Giang Province, Vietnam, are completely identical to that from Cambodia but slightly

different from that of Japan. This variation may be due to geographical distance and hosts. The Vietnamese and Cambodian samples were geographically closer and were collected from domestic pigs, whereas that from Japan was geographically far distant and was collected from wild boar.

S. ransomi VN1 S. ransomi VN2 S. ransomi VN3 60

S. ransomi VN4 S. ransomi VN5 KU724126 S. ransomi KH

61

AB453327 S. ransomi JP AB923887 S. venezuelens JP AB453330 S. venezuelensis JP

50

AB453323 S. papillosus JP 85 EF066361 S. papillosus DE

AB923886 S. papillosus JP 49 AB677955 S. fuelleborni JP

50

AB272235 S. fuelleborni JP 96 AB923889 S. ratti JP

U81581 S. ratti US 51

AB453315 S. stercoralis JP

49

AF279916 S. stercoralis UK 77 AB923888 S. stercoralis MM

AB205054 S. procyonis JP AB272231 S. callosciureus JP AB272232 S. robustus US AB923885 P. trichosuri JP

0.01

Figure 2. The molecular phylogenetic tree reconstructed using the Maximum Likelihood method

In comparison with other Strongyloides species, S. ransomi is genetically close to S. venezuelensis, although its morphological features are similar to S. papillosus (table 1; Schwartz & Allicata, 1930; Basir, 1950; Little, 1966). The position of S. papillosus in the phylogenetic tree is

unclear. While one each sequence of this species from Japan and Germany is completely identical with each other to make a distinct group, the other sequence (AB453323) from Japan shows a far distance from the earlier sequences, but is similar to and is clustered with S. venezuelensis group. This suggests 273

Morphological and molecular characterisation of Strongyloides ransomi

possibilities of various lineages of S. papillosus or abnormal sequence (AB453323). More sequences of S. papillosus from different

geographical locations should be analyzed to clarify this point.

Table 1. Morphometric comparison of S. ransomi, S. papillosus and S. venezuelensis Measurement Body length (mm) Body width (µm) Length of esophagus (mm) Vulva to anterior end (mm) Anus to posterior end (µm) Reference

S. ransomi 3.3 - 4.5 54 - 62 0.61 - 0.88 1.9 - 2.9 53 - 83 Schwartz & Allicata, 1930

Conclusion The Strongyloides adult worms collected from pigs in Bac Giang Province, Vietnam, were morphologically and molecularly identified as S. ransomi. The HVR-I of 18S rDNA sequence of S. ransomi from Vietnam was completely identical with that from Cambodia and was slightly different from that of Japan. In comparision to other Strongyloides species, S. ransomi is morphologically similar to S. papillosus, but, on 18S rDNA sequencebased molecular analysis, it is close to S. venezuelensis. REFERENCES

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S. papillosus 4.8 - 5.8 42 - 78 0.72 - 0.95 2.8 - 3.5 54 - 78 Basir, 1950

S. venezuelensis 2.0 - 3.2 33 - 41 0.60 - 0.78 1.4 - 2.2 38 - 58 Little, 1966

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