Abdel Rahman Beni-Suef University Journal of Basic and Applied Sciences Beni-Suef University Journal of (2020) 9:17 https://doi.org/10.1186/s43088-020-00046-y Basic and Applied Sciences RESEARCH Open Access Morphological and molecular characterization of Setaria equina in donkeys Mona Mohammed I. Abdel Rahman Abstract Background: Adult worms of Setaria equina mainly found in the peritoneal cavity of equine. They were nonpathogenic but might induce varied degrees of peritonitis and might migrate to the eye, brain, lung, and scrotum causing lacrimation, blindness, paraplegia, locomotor, and neurological disturbances. Identification by light microscopy is insufficient to differentiate Setaria species, and so scanning electron microscopy (SEM) is required to observe their ultrastructures. The study was performed on 80 donkeys from May 2018 to January 2019 for the detection of microfilaria in blood and the adult worms in the peritoneal cavity. The blood samples were either stained with Giemsa stain or examined by modified Knott’s technique for the detection of microfilariae. Adult worms were morphologically characterized based on light microscope and scanning electron microscopy (SEM). PCR was performed targeting the 12S rRNA gene followed by sequencing and phylogenetic analysis. Results: The current study recorded 21.6% and 16.2% prevalence rates for adult worms and microfilariae, respectively. By using SEM, this study was able to clarify the detailed structure of amphids, predeirids, vulva, arrangement, and number of male caudal papillae. PCR amplified products for 12S rRNA gene (408 bp) for adult worm and microfilaria. Sequence and phylogenetic analysis revealed that S. equina isolated in the current study from donkeys in Egypt (accession no., MH345965) shared 100% identity with isolates from horse and man in Italy and Iran, respectively and clustered in the same clade with S. digitata, S. tundra and S. labiatopapillosa. Conclusions: Identification with light microscopy lacked the ability to characterize different Setaria species, and so using scanning electron microscopy is considered a good choice to distinguish the ultrastructures. In addition, performing the phylogenetic analysis was necessary to detect relationships between different filarial worms, which could not detect by the morphological characterization of adult worms. Keywords: Equine, Microfilaria, Setaria equina, 12S rRNA, Ultrastructure 1 Background prenatal infection reported as a route of transmission for Setaria equina (S. equina) is a common vector borne Setaria species [2, 3]. pathogen of equines all over the world, especially in Adult worms mainly found in the peritoneal cavity of tropical zones. S. equina transmitted by Aedes aegypti horse and donkey. The worms were nonpathogenic but and Culex pipens where L1 developed to L3 within 2 might induce varied degrees of peritonitis and might mi- weeks in their thoracic muscles. Then, the equines ac- grate to the eye, brain, lung, and scrotum of equines quired the infection during mosquito’s blood meal and causing lacrimation, blindness, paraplegia, locomotor, the life cycle completed within 8-10 months [1]. Also, and neurological disturbances [4, 5]. Not only S. equina induced such pathogenic effects but also other Setaria Correspondence: [email protected]; [email protected] species infect cattle (S. digitata and S.cervi) could induce Department of Parasitology, Faculty of Veterinary Medicine, Zagazig blindness and CNS damage in equine where [6] recorded University, 1 Alzeraa Street, Zagazig City, Sharkia Province 44511, Egypt © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Abdel Rahman Beni-Suef University Journal of Basic and Applied Sciences (2020) 9:17 Page 2 of 8 horse blindness with S. digitata in Korea. Also, Abu El- dehydrated in graded ethanol, mounted over the stubs, Magd and Ahmed, Marzok and Desouky [3, 7] reported coated with gold coat, and examined with Quanta FEG250 the aberrant parasitism of adult worms in eye of donkey scanning electron microscope, operated at 20 KV in Na- in Egypt. Also, Nabie et al. and Taylor et al. [8, 9] stated tional Research Center, Dokki, Egypt [5, 18]. the zoonotic importance of S. equina in man. Many species of Setaria were reported all over the 2.2.2 Blood samples world, but S.equina considered the most popular species Ethylene Diamine Tetraacetic acid (EDTA) mixed blood recorded in donkeys. In Egypt, S.equina had been studied samples were collected, stained with Giemsa stain, or ex- – by many authors in different Governorates [3, 7, 10 13]. amined by modified Knott’s technique for detection of The highest rate was recorded in Benha Governorate [14]. microfilariae according to [19]. The diagnosis of microfilariasis depending upon the no- ticed clinical symptoms or serological tests is inaccurate, 2.3 DNA extraction and PCR amplification lacking the specificity and consuming time. As it is known DNA was extracted from adult worms and blood sam- that the DNA is stable in the life cycle stages of the parasite. ples using the QIAamp DNA Mini kit (Qiagen, Therefore, the PCR technique was performed in the current Germany, GmbH), and subjected to PCR targeting the study to diagnose microfilariasis using the extracted DNA 12S ribosomal RNA gene. PCR reaction was carried out from adult worms and microfilariae of S.equina. In Egypt, in a 0.2 tube containing 1.5 μl Max PCR Master Mix many previous studies had used the adult worms of S.equina (Takara, Japan), 0.25 μl of each primer (Bio Basic Canada in PCR, but this study is the first of its kind that used both of Inc.), 5 μl of DNA template, and up to 25 μl nuclease adult worms and their corresponding microfilariae in PCR. free water. The PCR cycling program and primers se- Also, the previous studies had been characterized S.equina in quences are listed in Table 1 [20]. horses, but fewer studies were known about donkeys. So the present study aimed to assess the following points: (a) investigate the prevalence rate of S. equina in donkeys in 2.4 Phylogenetic analysis Egypt, (b) diagnose the microfilariae early in blood sam- PCR products were purified by QIA quick PCR purifica- ples of infected donkeys, (c) identify the morphological tion kit (Qiagen, Valencia) and sequenced by Big dye features of the genus Setaria under the light microscope, Terminator V3.1 cycle sequencing kit (Perkin-Elmer). (d) characterize and differentiate the detailed ultrastruc- DNA sequences were obtained by Applied Biosystems tures of the species equina from others like digitata or 3130 genetic analyzer (HITACHI, Japan) and a BLAST® marsalli by scanning electron microscopy, and (e) detect analysis (Basic Local Alignment Search Tool). The se- the phylogenetic relationship between S. equina and other quence analysis was performed by the MegAlign module members of Filarioidea depended upon 12S rRNA gene. of Lasergene DNAStar [21] and the phylogenetic tree generated by using maximum likelihood, neighbor join- 2 Methods ing, and maximum parsimony in MEGA 6[22]. The 12S 2.1 Animals rRNA gene sequences generated in this study was depos- A total of 80 donkeys (10-30 years old) were examined ited in the GenBank under accession no. MH345965. during the period from May 2018 to January 2019 in Giza zoo, Dokki, Egypt. 3 Results 3.1 Morphological characterization 2.2 Sample collection and processing 3.1.1 By naked eye 2.2.1 Adult worms Adult worms inside the peritoneum of donkeys appeared Adult worms were collected from peritoneal cavity of don- milky white and thread like. Their measurements keys at the time of necropsy, washed with saline, cleared reached 45-70 mm (57 ± 2) long × 0.4-0.6 mm (0.45 ± with lactophenol, and identified under light microscope 0.02) wide in males with coiled end and reached 60-160 according to [15–17]. For the SEM, the adult worms were mm (110 ± 5) long × 0.6-0.91 mm (0.60 ± 0.04) wide in fixed in 2.5% buffered gluteraldehyde (pH 7.2) for 24 h, females with loose spiral end (Fig. 1a, b). The prevalence Table 1 PCR cycling program and primers sequences Primer Sequence 5"–3" Start Stop Product 1st De. Amplification F. Ex. Reference length 2nd De. An. Ex. 12SF 5′-GTT CCA GAA TAA TCG GCT A-3′ 7484 7502 408 bp 94 °C 5 min 94 °C 1 min 50 °C 1 min 72 °C 1 min 72 °C 10 min [20] 12SR 5′-ATT GAC GGA TG(AG) TTT GTA CC-3 7994 7975 40 cycles An. annealing, De. denaturation, F. Ex. final extension Abdel Rahman Beni-Suef University Journal of Basic and Applied Sciences (2020) 9:17 Page 3 of 8 Fig. 1 Photos and microphotos of Setaria equina adult worm and its developmental stages. a1 Adult S. equina in peritoneum of donkey (arrow). b1 Male (m) and female (f) worms. c1 Embryonated egg (× 100). d1 Egg with developing juvenile (J1) in coiled position (× 100). e1 Egg contained fully extended J1(× 100). f1 Free, full extended and sheathed J1(× 100). g1, h1 Microfilaria in Knott’s technique (× 100).