Original Article

Comparative Morphologic and Morphometric Study on the Developmental Aspects of In Vitro and In Vivo Reared Echinococcus granulosus Sensu Stricto Using Differential Interference Contrast (DIC)/Nomarski and Phase Contrast Microscopy

Abstract

Background: Echinococcus granulosus is a zoonotic parasite with worldwide distribution. The present study focused on comparative morphologic and morphometric observations on the developmental aspects of whole body, more special the reproductive structures of in vitro reared adult worms (RAW) and in vivo reared adult worms in definitive host (AWIDH) using differential interference contrast (DIC)/Nomarski, phase contrast and routine optical microscopy.

Methods: A total number of 10 in vitro and 10 in vivo reared adult worms of E. granulosus sensu stricto, G1 strain were selected. The worms were processed by Formaldehyde-Alcohol-Azocarmine-Lactophenol (FAAL). The details of morphological factors and reproductive structures of each worm including 25 biometrical parameters were studied by routine optical, phase contrast and Nomarski microscopy. The details of the samples were photographed, measured and analyzed. The fine structures of the parasite including the details of cirrus sac and developmental stages in different strobila were more obvious observing by Nomarski microscopy. 

Results: The morphometric characters in the RAW and AWIDH showed that length of immature proglottid, length of mature proglottid, length of suckers are larger in RAW than AWIDH worms with statistical difference. Characters in E. granulosus of RAW and AWIDH showed that total number of segments, number of mature segments and the total number of testes were greater in RAW than AWIDH worms; while only the number of mature segments was statistically different is two groups.

Conclusion: Application of DIC/Nomarski and phase contrast microscopy together with morphometric criteria are useful means for comparing the developmental aspects of in vitro and in vivo reared adults of E. granulosus.

Deplazes P, Rinaldi L, Alvarez Rojas CA, Torgerson PR, Harandi MF, Romig T,Antolova D, Schurer JM, Lahmar S, Cringoli G, Magambo J, Thompson RC, Jenkins EJ. Global Distribution of Alveolar and Cystic Echinococcosis. Adv Parasitol. 2017; 95:315-493.

Rokni, MB. Echinococcosis /hydatidosis in Iran. Iranian J Parasitol: 2009; 4(2): 1-16.

Thompson RCA, Deplazes P, Lumbery A. Preface. Adv Parasitol. 2017; 96: xiii-xiv.

Smyth JD and McManus DP. The Physiology and Biochemistry of Cestodes. Cambridge University Press. 2007.

Eckert J and Deplazes P. Biological, Epidemiological, and Clinical Aspects of Echinococcosis, a Zoonosis of Increasing Concern. Clin Microbiol Rev. 2004; 17(1):107-135.

Ito A. Review of "Echinococcus and Echinococcosis, Part A." edited by R. C. Andrew Thompson, Alan J. Lymbery and Peter Deplazes. Parasit Vectors. 2017; 10(1):408.

Agudelo Higuita NI, Brunetti E, McCloskey C. Cystic Echinococcosis. J Clin Microbiol. 2016; 54(3):518-523.

Neumayr A, Tamarozzi F, Goblirsch S, Blum J, Brunetti E. Spinal cystic echinococcosis--a systematic analysis and review of the literature: part 1. Epidemiology and anatomy. PLoS Negl Trop Dis. 2013; 7(9):2450.

Brunetti E, Garcia HH, Junghanss T. On behalf of the members of the International CE Workshop in Lima, Peru, 2009. Cystic Echinococcosis: Chronic, Complex, and Still Neglected. PLoS Negl Trop Dis. 2011; 5(7):e1146.

Thompson RCA and Lymbery AJ. Let’s not forget the thinkers. Trends Parasitol. 2013; 29:581-584.

Li J, Wu C, Wang H, Liu H, Vuitton DA, Wen H, Zhang W. Boiling sheep liver or lung for 30 minutes is necessary and sufficient to kill Echinococcus granulosus protoscoleces in hydatid cysts. Parasite. 2014; 21:64.

Eslami A and Hosseini SH. Echinococcus granulosus infection of farm dogs of Iran. Parasitol Res. 1998; 84(3):205-207.

Sadjjadi SM, Mikaeili F, Karamian M, Maraghi S, Sadjjadi FS, Shariat-Torghaban S, Kia EB. Evidence that the Echinococcus granulosus G6 genotype has an affinity for the brain in humans. Int J Parasitol. 2013; 43(11): 875-877.

Ebrahimipour M, Sadjjadi SM, Yousofi Darani H, Najjari M. Molecular Studies on Cystic Echinococcosis of Camel (Camelus dromedarius) and Report of Echinococcus ortleppi in Iran. Iran J Parasitol. 2017; 12(3):323-331.

Murphy DB. Differential interference contrast (DIC) microscopy and modulation contrast microscopy in fundamentals of light microscopy and digital imaging. Wiley-Liss, New York. 2001. 153.

Mok DZ, Sternberg PW, Inoue T. Morphologically defined sub-stages of C.elegans vulval development in the fourth larval stage. BMC Dev Biol. 2015; 12(15):26.

Salmon E and Tran P. High-resolution video-enhanced differential interference contrast (VEDIC) light microscope,” in Video Microscopy, G. Sluder and D. Wolf, eds. (Academic, New York). 2003.

Fard AM, Mahjoubfar A, Goda K, Gossett DR, Di Carlo D, Jalali B. Nomarski serial time-encoded amplified microscopy for high-speed contrast-enhanced imaging of transparent media. Biomed Opt Express. 2011; 2(12):3387-3392.

Smyth JD and Davies Z. In vitro culture of the strobilar stage of Echinococcus granulosus (sheep strain): A review of basic problems and results. Int J Parasitol. 1974; 4: 631-644.

Macpherson CN and Smyth JD. In vitro culture of the strobilar stage of Echinococcus granulosus from protoscoleces of human, camel, cattle, sheep and goat origin from Kenya and buffalo origin from India. Int J Parasitol. 1985; 15(2):137-40.

Mohammadzadeh T, Sadjjadi SM, Rahimi HM. Still and Moving Image Evidences for Mating of Echinococcus granulosus Reared in Culture Media. Iran J Parasitol. 2014; 9(1):129-33.

Soriano SV, Debiaggi MF, Pierangeli NB, Pianciola LA, Bergagna HFJ, Lazzarini LE, Mazzeo ML, Basualdo JA. First study about the development of adult Echinococcus canadensis G6 genotype of goat origin in experimentally infected dogs. Vet Parasitol. 2016; 228:6-12.

Koziol U. Evolutionary developmental biology (evo-devo) of cestodes. Exp Parasitol. 2017; 180:84-100.

Thompson RCA. Biology and Systematics of Echinococcus. Adv Parasitol. 2017; 95:65-109.

Smyth JD. Cestoda. In: Smyth JD. ( Ed.) In vitro Cultivation of Parasitic Helminthes. London: CRC press; 1990. p. 123-137.

Mohammadzadeh T, Sadjjadi SM, Rahimi HM, Shams S. Establishment of a modified in vitro cultivation of protoscoleces to adult Echinococcus granulosus; an important way for new investigations on hydatidosis. Iran J Parasitol. 2012; 7(1):59-66.

Zahabiun F, Sadjjadi SM, Esfandiari F. Development of a double glass mounting method using formaldehyde alcohol azocarmine lactophenol (FAAL) and its evaluation for permanent mounting of small nematodes. Iran J Parasitol. 2015; 10:617-624.

Yanagida T, Mohammadzadeh T, Kamhawi S, Nakao M, Sadjjadi SM, Hijjawi N, Abdel-Hafez SK, Sako Y, Okamoto M, Ito A. Genetic polymorphisms of Echinococcus granulosus sensu stricto in the Middle East. Parasitol Int. 2012; 61: 599-603.

Smyth JD, Miiller HJ, Howkins AB. Further analysis of the factors controlling strobilization, differentiation, and maturation of Echinococcus granulosus in vitro. Exp Parasitol. 1967; 21:31-41.

Smyth JD, Howkins AB, Barton M. Factors controlling the differentiation of the hydatid organism, Echinococcus granulosus, intocystic or strobilar stages in vitro. Nature. 1966; 211:1374-1377.

Rahimi HR, Mohammadzadeh T, Sadjjadi SM. Immune responses to antigens of in vitro reared Echinococcus granulosus adult worms in Balb/c mice. Iran J Immunol. 2011; 8(4):236-43.

Rahimi HR, Mohammadzadeh T, Sadjjadi SM, Sarkari B, Zahabiun F. BALB/c Mice Immunity to Hydatidosis Induced by In-vitro Reared Echinococcus granulosus Adult Worm Antigens. Iran J Immunol. 2017; 14(2):123-133.

Thompson RCA and Jenkins DJ. Echinococcus as a model system: Biology and epidemiology. Int J Parasitol. 2014; 44:865-877.

Smyth JD. Parasites as biological models. Parasitology. 1969; 59(1):73-91.

Koziol U and Brehm K. Recent advances in Echinococcus genomics and stem cell research. Vet Parasitol. 2015; 213(3-4): 92-102.

Macpherson CN and Smyth JD. In vitro culture of the strobilar stage of Echinococcus granulosus from protoscoleces of human, camel, cattle, sheep and goat origin from Kenya and buffalo origin from India. Int J Parasitol. 1985; 15(2):137-40.

Monteiro KM, De Carvalho MO, Zaha A, Ferreira HB. Proteomic analysis of the Echinococcus granulosus metacestode during infection of its intermediate host. Proteomics. 2010; 10:1985-1999.

Zheng H, Zhang W, Zhang L, Zhang Z, Li J, Lu G, Zhu Y, Wang Y, Huang Y, Liu J, Kang H, Chen J, Wang L, Chen A, Yu SH, Gao Z, Jin L, Gu W, Wang Z, Zhao L, Shi B, Wen H, Lin R, K Jones M, Brejova B, Vinar T, Zhao G, McManus DP, Chen Z, Zhou Y, Wang S. The genome of the hydatid tapeworm Echinococcus granulosus. Nat Genet. 2013; 45:1168-1175.

Dezaki ES, Yaghoubi MM, Spiliotis M, Boubaker G, Taheri E, Almani PG, Tohidi F, Harandi MF, Gottstein B. Comparison of ex vivo harvested and in vitro cultured materials from Echinococcus granulosus by measuring expression levels of five genes putatively involved in the development and maturation of adult worms. Parasitol Res. 2016; 115(11):4405-4416.

Thompson RCA, Deplazes P, Eckert J. Uniform strobilar development of Echinococcus multilocularis in vitro from protoscolex to immature stages. J Parasitol. 1990; 76(2):240-7.

Dubinsky P, Stefancikova A, Turcekova L, Macko Jk, Soltys J. Development and morphological variability of Echinococcus granulosus. Parasitol Res. 1998; 84(3):221-229.

Kumaratilake LM and Thompson RCA. Morphological characterisation of Australian strains of Echinococcus granulosus. Int J Parasitol. 1984; 14(5):467-77.

Hosseini SH and Eslami A. Morphological and developmental characteristics of Echinococcus granulosus derived from sheep, cattle and camels in Iran. J Helminthol. 1998; 72:337-341.

Kumaratilake LM, Thompson RCA, Dunsmore JD. Comparative strobilar development of Echinococcus granulosus of sheep origin from different geographical areas of Australia in vivo and in vitro. Int J Parasitol. 1983; 13(2):151-156.

Hussain A, Maqbool A, Tanveer A, Anees A. Studies on morphology of Echinococcus granulosus from different animal-dog origin. Punjab Univ J Zool 2005; 20(2):151-157.

Pandey VS. Observations on the morphology and biology of Echinococcus granulosus (Batsch, 1786) of goat-dog origin. J Helminthol. 1972; 46(3):219-33.

Smyth, J.D. Asexual and sexual differentiation in cestodes: especially Mesocestoides and Echinococcus. MacInnis, A.J. (Ed.) Molecular paradigms for eradicating helminthic parasites. New York, Alan R. Liss. 1987. pp. 19–33.

Howell MJ and Smyth JD. Maintenanceand cultivation of Echinicoccus species in vivo and in vitro in: Thompson RCA, Lymbery AJ. Echinococcus and hydatid disease Wallingford. CAB International Wallingford, Oxon, UK 1995; 200-232.

Thompson RCA, Boxell AC, Ralston BJ, Constantine CC, Hobbs RP, Shury T, Olson ME. Molecular and morphological characterization of Echinococcus in cervids from North America. Parasitology. 2006; 132:439-447.

Lymbery AJ, Jenkins EJ, Schurer JM, Thompson RCA. Echinococcus canadensis, E. borealis, and E. intermedius. What's in a name? Trends Parasitol. 2015; 31(1):23-29.

Files
IssueVol 14 No 4 (2019) QRcode
SectionOriginal Article(s)
DOI https://doi.org/10.18502/ijpa.v14i4.2093
Keywords
Echinococcus granulosus sensu stricto G1 strain DIC/Nomarski microscopy Phase contrast Morphology Morphometry

Rights and permissions
Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
How to Cite
1.
SADJJADI SF, MOTAMEDI M, MOHAMMADZADEH T, SADJJADI SM. Comparative Morphologic and Morphometric Study on the Developmental Aspects of In Vitro and In Vivo Reared Echinococcus granulosus Sensu Stricto Using Differential Interference Contrast (DIC)/Nomarski and Phase Contrast Microscopy. Iran J Parasitol. 2019;14(4):510-520.