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Molecular Characterization of Setaria equina Infecting Donkeys (Equus asinus) from Egypt



Ibrahim Abbas, Moustafa Al-Araby and Yara Al-Kappany
 
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ABSTRACT

Objective: Since few molecular data about Setaria equina are available, as this study proposed in order to investigate the molecular characteristics and the phylogenetic position of S. equina isolates from donkeys which was previously studied in horses. Methodology: The DNA was extracted from adult worms of S. equina found in the peritoneal cavity of donkeys slaughtered at Giza Zoo Abattoir, Egypt. A fragment length of the cytochrome oxidase subunit 1 (COX 1) gene (683 bp) was amplified using PCR. Purified PCR product was sequenced. Sequences were aligned with those published on GenBank and subsequently, the phylogenetic tree was constructed. Results: Results of the BLAST search showed that our isolates from donkeys are homologous (99% identity) with that from horses (AJ544873). Phylogenetic analysis exhibited the sister relationship between S. equina isolates from both donkeys and horses which illustrates the conspecificity between them and suggesting the cross transmission of this parasite species among different equids. Conclusion: The genetic relevance of this parasite to the other filarial worms was discussed in details. This is the first report about the molecular identification of S. equina infecting donkeys from Egypt.

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  How to cite this article:

Ibrahim Abbas, Moustafa Al-Araby and Yara Al-Kappany, 2016. Molecular Characterization of Setaria equina Infecting Donkeys (Equus asinus) from Egypt. Research Journal of Parasitology, 11: 73-78.

DOI: 10.3923/jp.2016.73.78

URL: https://scialert.net/abstract/?doi=jp.2016.73.78
 
Received: May 13, 2016; Accepted: May 30, 2016; Published: September 15, 2016



INTRODUCTION

Setaria equina (family: Onchocercidae, subfamily: Setariinae) is a nematode filarial parasite commonly found in the peritoneal cavity of equines in different geographical regions of the world. The infection is believed to be transmitted by Aedes or Culex mosquitoes (Levine, 1985; Arundel, 1978; Coleman et al., 1985). The parasite is might erratically incriminated in the eye, brain, spinal medulla, vaginal sac and testicles of horses (Soulsby, 1982; Rodriguez-Vivas et al., 2000; Yadav et al., 2006; Gangwar et al., 2008; Kornas et al., 2010). Although infections are typically benign, pathologies of the eye and central nervous system (cerebrospinal nematodiasis) have been attributed to S. equina (Frauenfelder et al., 1980). Moreover, equine testicular lesions were noted (Marino et al., 2009). Taylor et al. (2001) recorded S. equina as one of the zoonotic species causing human disease.

The adult worms are thread-like ranged from 5-13 cm in length with the males being the smaller (Levine, 1985). The sheathed microfilaria worms are approximately 250-269 μm and found in the blood (Yeargan et al., 2009). Few studies are concerned with the prevalence of S. equina in horse populations. Low incidences were reported, 2% (Mfitilodze and Hutchinson, 1989), 4.4% (Al Anazi and Alyousif, 2011) and 8% (Gawor, 1995), while moderate prevalence rate (15%) was noted in Turkey (Oge et al., 2003). In Egypt, the infection with Setaria equina has been recorded (Abu El-Magd and Ahmed, 1994; Abdel-Wahab and Ashour, 1999; Marzok and Desouky, 2009). Still in Egypt, a high prevalence rate (40.08%) was reported in donkeys (Ahmed et al., 2011).

Nearly, 43 species of the genus Setaria have been identified so far in the world. The morphological characters alone are insufficient to establish the phylogenetic relationships and position of filarial nematodes (Chabaud and Bain, 1994), due to their similar morphological characteristics (Yatawara et al., 2007). Phylogenetic analysis is needed to evaluate and confirm the morphological description and taxonomy of Setaria species. It was reported that S. digitata and S. labiatopapillosa appeared to be sisters, as do S. equina and S. tundra (Yatawara et al., 2007). While, Alasaad et al. (2012) add S. cervi to the group of S. digitata and S. labiatopapillosa based on COX 1 sequence.

Setaria equina infecting horses (Equus caballus) from Italy was studied molecularly (Casiraghi et al., 2004). An important point of debate is that if S. equina infecting horses and those infecting donkeys are conspecific. As well as it is known that no molecular studies have been carried out to characterize this parasite species from donkeys and to determine its phylogenetic relationship with the other filarial worms.

This study was planned in order to find the molecular characteristics and the phylogenetic position of S. equina infecting donkeys, slaughtered at Giza Zoo Abattoir, Egypt, based on partial sequences of the mitochondrial cytochrome c oxidase subunit 1 (COX 1) gene. This study may help in future epidemiological prospects for developing control regimens against S. equina.

MATERIALS AND METHODS

Samples collection: Three adult nematode worms were collected during peritoneal cavities inspection of the slaughtered donkeys at Giza Zoo Abattoir, Egypt. These worms were identified as S. equina according to their morphological characteristics (Soulsby, 1982). Nematodes were washed 3 times by phosphate buffer saline and preserved in ethanol 70% until the DNA extraction.

DNA extraction: Genomic DNA was extracted from each worm using the standard phenol/chloroform technique (Sambrook et al., 1989).

PCR amplification: The PCR amplification of a fragment of the COX 1 gene was carried out in 35 μL final PCR mixture contained 2 μL of template DNA, 1 μL (25 μM) of each primer (Casiraghi et al., 2001) COX 1 intF (5'-TGATTGGTGGTTTTGGTAA-3') and COX 1 intR (5'-ATAAGTACGAGTATCAATATC-3'), 0.7 μL (10 mM) dNTP mix, 3.5 μL of taq buffer (10x), 0.35 μL Taq polymerase (5Prime Perfect TaqTm) and 26.45 μL nuclease free water. Negative control with no DNA was used. For amplification, samples were subjected to the following thermal profile, initial denaturation (94°C for 4 min) followed by 30 cycles of each of denaturation (94°C for 1 min), annealing (52°C for 1 min) and extension (72°C for 50 sec), then a final extension step (72°C for 5 min).

The resulted PCR products were subjected to gel electrophoresis using 1% agarose gel stained with ethidium bromide. Bands on gel nearly at 680 bp were purified with QIA quick PCR purification column (QIAGEN, GmbH, Hilden, Germany), then commercially sequenced using the PCR primers as sequencing primer. Searching for sequence similarity of samples was done using NCBI BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The obtained sequences were aligned with those published on the GenBank (Table 1) and the phylogenetic tree was constructed using the software Mega (version 6).

Table 1: Retrieved COX 1 sequences from GenBank which used for the phylogenetic tree construction

RESULTS

For all the three examined S. equina samples from donkeys, PCR amplification of a fragment of the COX 1 gene resulted in gel bands at 680 bp length, while the negative controls gave no bands.

Sequence polymorphism: Sequences from the three investigated S. equina worms were identical. Results of the BLAST search showed a 99% identity between the revealed S. equina from donkeys in this study and those from horses in Italy which deposited in GenBank under the accession No. AJ544873 with 7 nucleotide substitution C50T, A61T, T104C, C202G, A289G, A564T and A577G (Fig. 1). A deletion was noted at the site 592 of donkeys’ S. equina sequence. Concerning the relationship with the other Setaria species, there were nearly similar identity percents between their sequences and those reported in this study. A 90% identity was noted with S. tundra (AM749298), S. labitopapillosa (AJ544872) and S. cervi (JF800924), while 89% identity was found with S. digitata (EF174426).

Phylogenetic analysis: As shown in Fig. 2, data emerged from the phylogenetic tree based on COX 1 sequences showed that the genus Setaria shared the same calde with Onchocerca and Dirofilaria, while the Wuchereria, Brugia and Loa loa formed separate clades. Moreover, Setaria species are monophyletic and localized in 2 sister groups, the first including S. equina and S. tundra while, the other group consisted of S. digitata, S. cervi and S. labitopapillosa. The revealed S. equina from donkeys in the present study was found in the same sister group with S. equina recovered from horses.

DISCUSSION

Filarioid nematodes affect millions of people and animals allover the world bringing up to major health hazards and economic losses (World Health Organization, 2006).

Studying the molecular characteristics of filarial worms is important to emphasis their identification and taxonomy since their similar morphological characteristics weakened the evolutionary pattern (Yatawara et al., 2007). Another considerable point for studying the genetic relationship between the filarioid worms is the cross antigenicity between them. Antigens from Setaria species have the potential for immunodiagnosis of human filariosis. Cross reaction was found between S. equina antigens and antibodies in the sera of Wuchereria bancrofti infected patients, especially in the chronic infected subjects (Bahgat et al., 2011).

Like what have been previously stated by Casiraghi et al. (2004) and Yatawara et al. (2007), these results showed that the filarial worms formed 2 main clades according to their phylogenetic position, the genus Setaria is a member of a large clade along with Onchocerca and Dirofilaria, while, Wuchereri bancrofti, Brugia malayi and Loa loa are located in a separate clades. Moreover, both Wuschereria bancrofti and Brugia malayi are appeared to be sisters.

Fig. 1:
Alignment of the partial COX 1 nucleotide sequence of Setaria equina isolated from donkey with that from horse (AJ544873). Seven nucleotide substitutions are noted: C50T, A61T, T104C, C202G, A289G, A564T and A577G. There is a deletion at the site 592 in the donkey`s sequence

Fig. 2:
Phylogenetic tree inferred from partial COX 1 nucleotide sequences using the neighbor joining method in the software Mega 6 and showing a sister relationship between isolates of Setaria equina from both donkeys and horses. Scale bar indicates the proportion of sites changing along each branch

In this study, Dirofilaria does not share the same branch with Setaria, opposite to what have been stated by Jayasinghe and Wijesundera (2003).

Concerning the genus Setaria, the inferred data from this study confirmed the results of the phylogenetic position within Setaria species which previously recorded (Casiraghi et al., 2004; Yatawara et al., 2007; Alasaad et al., 2012). All Setaria species are found in the same phyletic group in which S. equina and S. tundra appeared to be sisters, as do S. digitata, S. cervi and S. labitopapillosa.

Setaria equina is reported globally. The molecular characteristics and phylogenetic position of S. equina was firstly described by Casiraghi et al. (2004). They collected the samples from horses in Italy. This study is the first report about the molecular characterization of S. equina infecting donkeys. Results showed that both of the revealed S. equina isolates either from horses or donkeys are sisters and this confirmed the conspeicificty between them and in turn, this species could be crossly transmitted between horses and donkeys.

CONCLUSION

In conclusion, a further study with frequent number of S. equina specimens from different equides (horses, donkeys and mules) as well as the arthropode vectors should be carried out in order to enhance the understanding about the transmission of this parasite and its relation with the other members of the genus Setaria and with the other filarial worms.

REFERENCES
1:  Abdel-Wahab, T.M. and A.A. Ashour, 1999. Scanning electron microscopy of the two filarial nematodes Setaria equina and Onchocerca cervicalis from kafr El-Sheikh area, Egypt. Alex. J. Vet. Sci., 15: 541-547.

2:  Abu El-Magd, A. and Z.G. Ahmed, 1994. The occurance of Setaria equina in donkeys eyes and their treatment. Assiut Vet. Med. J., 31: 86-90.

3:  Ahmed, N.E., L.M. El-Akabawy, M.Y. Ramadan and A.M.M. Radwan, 2011. Studies on helminthe parasites in necropsied donkeys in Egypt. Benha Vet. Med. J., 1: 153-162.
Direct Link  |  

4:  Al Anazi, A.D. and M.S. Alyousif, 2011. Prevalence of non-strongyle gastrointestinal parasites of horses in Riyadh region of Saudi Arabia. Saudi J. Biol. Sci., 18: 299-303.
CrossRef  |  Direct Link  |  

5:  Alasaad, S., I. Pascucci, M.J. Jowers, R.C. Soriguer, X.Q. Zhu and L. Rossi, 2012. Phylogenetic study of Setaria cervi based on mitochondrial cox1 gene sequences. Parasitol. Res., 110: 281-285.
CrossRef  |  Direct Link  |  

6:  Arundel, J.H., 1978. Parasitic disease of the horse. Veterinary Review No. 18, University of Sydney Post Graduate Foundation in Veterinar, Sydney, pp: 1-83.

7:  Bahgat, M.M., A.H. Saad, G.A. El-Shahawi, A.M. Gad, R.M. Ramzy, A. Ruppel and A. Abdel-Latif, 2011. Cross-reaction of antigen preparations from adult and larval stages of the parasite Setaria equina with sera from infected humans with Wuchereria bancrofti. Eastern Mediterr. Health J., 17: 679-686.
Direct Link  |  

8:  Casiraghi, M., O. Bain, R. Guerrero, C. Martin and V. Pocacqua et al., 2004. Mapping the presence of Wolbachia pipientis on the phylogeny of filarial nematodes: Evidence for symbiont loss during evolution. Int. J. Parasitol., 34: 191-203.
CrossRef  |  Direct Link  |  

9:  Casiraghi, M., T.J.C. Anderson, C. Bandi, C. Bazzocchi and C. Genchi, 2001. A phylogenetic analysis of filarial nematodes: Comparison with the phylogeny of Wolbachia endosymbionts. Parasitology, 122: 93-103.
CrossRef  |  Direct Link  |  

10:  Chabaud, A.G. and O. Bain, 1994. The evolutionary expansion of the Spirurida. Int. J. Parasitol., 24: 1179-1201.
CrossRef  |  Direct Link  |  

11:  Coleman, S.U., T.R. Klei and D.D. French, 1985. Prevalence of Setaria equina (Nematode: Onchocercidae) in Southeastern Louisiana horses. J. Parasitol., 71: 512-513.
PubMed  |  Direct Link  |  

12:  Eisenbarth, A., D. Ekale, J. Hildebrandt, M.D. Achukwi, A. Streit and A. Renz, 2013. Molecular evidence of Siisa form, a new genotype related to Onchocerca ochengi in cattle from North Cameroon. Acta Tropica, 127: 261-265.
CrossRef  |  Direct Link  |  

13:  Ferri, E., M. Barbuto, O. Bain, A. Galimberti and S. Uni et al., 2009. Integrated taxonomy: Traditional approach and DNA barcoding for the identification of filarioid worms and related parasites (Nematoda). Front. Zool., Vol. 6. 10.1186/1742-9994-6-1

14:  Frauenfelder, H.C., K.R. Kazacos and J.R. Lichtenfels, 1980. Cerebrospinal nematodiasis caused by a filariid in a horse. J. Am. Vet. Med. Assoc., 177: 359-362.
PubMed  |  

15:  Fukuda, M., Y. Otsuka, S. Uni, O. Bain and H. Takaoka, 2010. Genetic evidence for the presence of two species of Onchocerca from the wild boar in Japan. Parasite, 17: 33-37.
CrossRef  |  Direct Link  |  

16:  Gangwar, A.K., S. Devi, H.N. Singh and A. Singh, 2008. Ocular filariasis in equines. Indian Vet. J., 85: 547-548.

17:  Gawor, J.J., 1995. The prevalence and abundance of internal parasites in working horses autopsied in Poland. Vet. Parasitol., 58: 99-108.
CrossRef  |  PubMed  |  Direct Link  |  

18:  Ghedin, E., S. Wang, D. Spiro, E. Caler and Q. Zhao et al., 2007. Draft genome of the filarial nematode parasite Brugia malayi. Science, 317: 1756-1760.
CrossRef  |  Direct Link  |  

19:  Jayasinghe, D.R. and W.S.S. Wijesundera, 2003. Differentiation of Setaria digitata and Setaria labiatopapillosa using molecular markers. Vet. J., 165: 136-142.
CrossRef  |  Direct Link  |  

20:  Kornas, S., M. Pozor, A. Okolski and B. Nowosad, 2010. [The case of the nematode Setaria equina found in the vaginal sac of the stallion's scrotum]. WiadomoSci parazytologiczne, 56: 319-321, (In Polish).
PubMed  |  Direct Link  |  

21:  Kronefeld, M., H. Kampen, R. Sassnau and D. Werner, 2014. Molecular detection of Dirofilaria immitis, Dirofilaria repens and Setaria tundra in mosquitoes from Germany. Parasit. Vectors, Vol. 7. 10.1186/1756-3305-7-30

22:  Levine, N.D., 1985. Nematode Parasites of Domestic Animals and of Man. Burgess Publishing Co., Minneapolis, pp: 477.

23:  Marino, G., A. Zanghi, M. Quartuccio, S. Cristarella, M. Giuseppe and G. Catoneb, 2009. Equine testicular lesions related to invasion by nematodes. J. Equine Vet. Sci., 29: 728-733.
CrossRef  |  Direct Link  |  

24:  Marzok, M.A. and A.R. Desouky, 2009. Ocular infection of donkeys (Equus asinus) with Setaria equina. Trop. Anim. Health Prod., 41: 859-863.
CrossRef  |  Direct Link  |  

25:  McNulty, S.N., A.S. Mullin, J.A. Vaughan, V.V. Tkach, G.J. Weil and P.U. Fischer, 2012. Comparing the mitochondrial genomes of Wolbachia-dependent and independent filarial nematode species. BMC Genom., Vol. 13. 10.1186/1471-2164-13-145

26:  Mfitilodze, M.W. and G.W. Hutchinson, 1989. Prevalence and intensity of non-strongyle intestinal parasites of horses in Northern Queensland. Aust. Vet. J., 66: 23-26.
CrossRef  |  Direct Link  |  

27:  Oge, S., H. Oge, A. Yildirim and F. Kircali, 2003. Setaria equina infection of Turkish equines: Estimates of prevalence based on necropsy and the detection of microfilaraemia. Ann. Trop. Med. Parasitol., 97: 403-409.
CrossRef  |  Direct Link  |  

28:  Ramesh, A., S.T. Small, Z.A. Kloos, J.W. Kazura, T.B. Nutman, D. Serre and P.A. Zimmerman, 2012. The complete mitochondrial genome sequence of the filarial nematode Wuchereria bancrofti from three geographic isolates provides evidence of complex demographic history. Mol. Biochem. Parasitol., 183: 32-41.
CrossRef  |  Direct Link  |  

29:  Sambrook, J., E.F. Fritsch and T.A. Maniatis, 1989. Molecular Cloning: A Laboratory Manual. 2nd Edn., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA., ISBN-13: 9780879695774, Pages: 397.

30:  Soulsby, E.J.L., 1982. Helminths, Arthropods and Protozoa of Domesticated Animals. 7th Edn., Bailliere Tindall, London, UK., ISBN: 9780702008207, pp: 316-319.

31:  Taylor, L.H., S.M. Latham and M.E.J. Woolhouse, 2001. Risk factors for human disease emergence. Philos. Trans. R. Soc. London B: Biol. Sci., 356: 983-989.
CrossRef  |  PubMed  |  Direct Link  |  

32:  Uni, S., M. Fukuda, T. Agatsuma, O. Bain and Y. Otsuka et al., 2015. Onchocerca takaokai n. sp. (Nematoda: Filarioidea) in Japanese wild boars (Sus scrofa leucomystax): Description and molecular identification of intradermal females. Parasitol. Int., 64: 493-502.
CrossRef  |  Direct Link  |  

33:  Rodriguez-Vivas, R.I., U. Dzul-Canche, E.M. Sierra-Lira and F.J. Aranda-Cirero, 2000. [Filariosis for Setaria equina in a horse from the state of Yucatan, Mexico]. Revista Biomedica, 11: 183-185, (In Spanish).
Direct Link  |  

34:  World Health Organization, 2006. Global programme to eliminate lymphatic filariasis: Annual report on lymphatic filariasis. Wkly. Epidemiol. Record., 82: 361-380.

35:  Yadav, A., A. Kumar, M.S. Bhadwal, J.K. Khajuria and A. Gupta, 2006. Ocular setariosis in horses: A case study. J. Vet. Parasitol., 20: 183-184.
Direct Link  |  

36:  Yatawara, L., S. Wickramasinghe, M. Nagataki, R.P.V.J. Rajapakse and T. Agatsuma, 2007. Molecular characterization and phylogenetic analysis of Setaria digitata of Sri Lanka based on CO1 and 12S rDNA genes. Vet. Parasitol., 148: 161-165.
CrossRef  |  Direct Link  |  

37:  Yeargan, M.R., E.T. Lyons, S.A. Kania, S. Patton, C.C. Breathnach, D.W. Horohov and D.K. Howe, 2009. Incidental isolation of Setaria equina microfilariae in preparations of equine peripheral blood mononuclear cells. Vet. Parasitol., 161: 142-145.
CrossRef  |  Direct Link  |  


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