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A Simple and Rapid Staining Technique for Sex Determination of Trichinella Larvae Parasites by Confocal Laser Scanning Microscopy

Part of: Micrographia Collection

Published online by Cambridge University Press:  28 October 2019

Inese Gavarane Open the ORCID record for Inese Gavarane [Opens in a new window] ,
Elena Kirilova ,
Ilze Rubeniņa ,
Ligita Mežaraupe ,
Sergejs Osipovs ,
Gunita Deksne ,
Aleksandrs Pučkins ,
Inese Kokina ,
Andrejs Bulanovs  and
Muza Kirjušina
Inese Gavarane*
Affiliation:
Institute of Life Sciences and Technology, Daugavpils University, Daugavpils LV-5401, Latvia
Elena Kirilova
Affiliation:
Institute of Life Sciences and Technology, Daugavpils University, Daugavpils LV-5401, Latvia
Ilze Rubeniņa
Affiliation:
Institute of Life Sciences and Technology, Daugavpils University, Daugavpils LV-5401, Latvia
Ligita Mežaraupe
Affiliation:
Institute of Life Sciences and Technology, Daugavpils University, Daugavpils LV-5401, Latvia
Sergejs Osipovs
Affiliation:
Institute of Life Sciences and Technology, Daugavpils University, Daugavpils LV-5401, Latvia
Gunita Deksne
Affiliation:
Institute of Food Safety, Animal Health and Environment BIOR, Riga LV-1076, Latvia Faculty of Biology, University of Latvia, Riga LV-1004, Latvia
Aleksandrs Pučkins
Affiliation:
Institute of Life Sciences and Technology, Daugavpils University, Daugavpils LV-5401, Latvia
Inese Kokina
Affiliation:
Institute of Life Sciences and Technology, Daugavpils University, Daugavpils LV-5401, Latvia
Andrejs Bulanovs
Affiliation:
Institute of Life Sciences and Technology, Daugavpils University, Daugavpils LV-5401, Latvia
Muza Kirjušina
Affiliation:
Institute of Life Sciences and Technology, Daugavpils University, Daugavpils LV-5401, Latvia
*
*Author for correspondence: Inese Gavarāne, E-mail: inese.gavarane@du.lv
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Abstract

The roundworms of Trichinella genus are worldwide distributed and their prevalence in nature is high. Trichinella genus parasites are the causative agents of foodborne zoonosis trichinellosis. The main prevention and control of the infection are meat inspection by the magnetic stirrer method for the detection of Trichinella larvae in muscle samples. The treatment can be effective if the parasite is discovered early in the intestinal phase. Once the Trichinella larva has reached the muscle tissue, the parasite remains therein and there is no treatment for this life cycle stage. The Trichinella species is dioecious with separate male and female individuals. The developed staining technique that uses confocal laser scanning microscopy (CLSM) displays sufficient results for Trichinella larvae examination and this protocol is applicable to study the internal and external structures and for the sex determination of T. britovi and T. spiralis larvae samples. In the present study, a luminescent derivative was synthesized and used for staining of T. spiralis and T. britovi larvae samples for the examination by CLSM. Various fixatives, such as AFA, 70% ethanol, and Bouin's and Carnoy's solutions were tested for sample preparation. The synthesized luminescent compound demonstrates best visualization results for samples fixed in Bouin's fixative.

Keywords

benzanthrone dyeBouin's fixativeconfocal laser scanning microscopyfixativesTrichinella larvae sex determination
Type
Micrographia
Information
Microscopy and Microanalysis , Volume 25 , Issue 6 , December 2019 , pp. 1491 - 1497
Copyright
Copyright © Microscopy Society of America 2019 

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References

Amos, WB, White, JG & Fordham, M (1987). Use of confocal imaging in the study of biological structures. Appl Opt 26, 3239–3243.Google Scholar
Ancell, H & Pires-daSilva, A (2017). Sex-specific lifespan and its evolution in nematodes. Semin Cell Dev Biol 70, 122–129.Google Scholar
Andy, JJ, O'Connell, JP, Daddario, RC & Roberts, WC (1977). Trichinosis causing extensive ventricular mural endocarditis with superimposed thrombosis. Evidence that severe eosinophilia damages endocardium. Am J Med 63, 824–829.Google Scholar
Bai, X, Wu, X, Wang, X, Guan, Z, Gao, F, Yu, J, Yu, L, Tang, B, Liu, X, Song, Y, Wang, X, Radu, B, Boireau, P, Wang, F & Liu, M (2012). Regulation of cytokine expression in murine macrophages stimulated by excretory/secretory products from Trichinella spiralis in vitro. Mol Cell Biochem 360, 79–88.Google Scholar
Bai, X, Wu, X, Wang, X, Liu, X, Song, Y, Gao, F, Miao, Y, Yu, L, Tang, B, Wang, X, Radu, B, Vallee, I, Boireau, P, Wang, F, Zhao, Y & Liu, M (2011). Inhibition of mammalian muscle differentiation by excretory secretory products of muscle larvae of Trichinella spiralis in vitro. Parasitol Res 110, 2481–2490.Google Scholar
Bone, LW & Shorey, HH (1978). Nematode sex-pheromones. J Chem Ecol 4, 595–612.Google Scholar
Bouwknegt, M, Devleesschauwer, B, Graham, H, Robertson, LJ, van der Giessen, JWB & the Euro-FBP Workshop Participants (2018). Prioritisation of food-borne parasites in Europe, 2016. Euro Surveill 23, 17-00161.Google Scholar
Bughdadi, FA (2010). Ultrastructural studies on the parasitic worm Trichinella spiralis. J Taibah Univ Sci 3, 33–38.Google Scholar
Davila, S, Manso, PPA, Bessa, ECA, Rodrigues, MLA & Dias, RJP (2010). Gross anatomy of the musculature and a new description of the reproductive system of Tanaisia bragai and Tanaisia inopina (Trematoda: Eucotylidae) analysed by confocal laser scanning microscopy. Acta Zool-Stocholm 91, 139–149.Google Scholar
Deksne, G, Segliņa, Z, Jahundoviča, I, Esīte, Z, Bakasejevs, E, Bagrade, G, Keidāne, D, Interisano, M, Marucci, G, Tonanzi, D, Pozio, E & Kirjušina, M (2016). High prevalence of Trichinella spp. in sylvatic carnivore mammals of Latvia. Vet Parasitol 231, 118–123.Google Scholar
Dobretsov, GE (1989). Fluorescent Probes in the Studies of Cells, Membranes and Lipoproteins. Moscow, Russia: Nauka.Google Scholar
Gonta, S, Utinans, M, Kirilov, G, Belyakov, S, Ivanova, I, Fleisher, M, Savenkov, V & Kirilova, E (2013). Fluorescent substituted amidines of benzanthrone: Synthesis, spectroscopy and quantum chemical calculations. Spectrochim Acta A 101, 325–334.Google Scholar
Gounaris, K, Smith, VP & Selkirk, ME (1996). Structural organisation and lipid composition of the epicuticular accessory layer of infective larvae of Trichinella spiralis. Biochim Biophys Acta 1281, 91–100.Google Scholar
Gozdzik, K, Odoevskaya, IM, Movsesyan, SO & Cabaj, W (2017). Molecular identification of Trichinella isolates from wildlife animals of the Russian Arctic territories. Helminthologia 54, 11–16.Google Scholar
Hao, Y, Zhao, X, Yang, J, Gu, Y, Sun, R & Zhu, X (2014). Monoclonal antibody targeting complement C9 binding domain of Trichinella spiralis paramyosin impairs the viability of Trichinella infective larvae in the presence of complement. Parasite Vector 7, 313–320.Google Scholar
Kapusta, P, Machalicky, O, Hrdina, R, Nepras, M, Zimmt, MB & Fidler, V (2003). Photophysics of 3-substituted benzanthrones: Substituent and solvent control of intersystem crossing. J Phys Chem A 107, 9740–9746.Google Scholar
Kirilova, E, Kecko, S, Mežaraupe, L, Gavarāne, I, Pučkins, A, Mickeviča, I, Rubeniņa, I, Osipovs, S, Bulanovs, A, Pupiņš, M & Kirjušina, M (2018 a). Novel luminescent dyes for confocal laser scanning microscopy used in Trematoda parasite diagnostics. Acta Biochim Pol 65, 449–454.Google Scholar
Kirilova, E, Mickevica, I, Mezaraupe, L, Puckins, A, Rubenina, I, Osipovs, S, Kokina, I, Bulanovs, A, Kirjusina, M & Gavarane, I (2019). Novel dye for detection of callus embryo by confocal laser scanning fluorescence microscopy. Luminescence 34, 353–359.Google Scholar
Kirilova, EM, Puckins, AI, Romanovska, E, Fleisher, M & Belyakov, SV (2018 b). Novel amidine derivatives of benzanthrone: Effect of bromine atom on the spectral parameters. Spectrochim Acta A 202, 41–49.Google Scholar
Kirjušina, M, Deksne, G, Marucci, G, Bakasejevs, E, Jahundoviča, I, Daukšte, A, Zdankovska, A, Bērziņa, Z, Esīte, Z, Bella, A, Galati, F, Krūmiņa, A & Pozio, E (2015). A 38-year study on Trichinella spp. in wild board (Sus scrofa) of Latvia shows a stable incidence with an increased parasite biomass in the last decade. Parasite Vector 8, 1–8.Google Scholar
Kozek, WJ (1975). Trichinella spiralis: Morphological characteristics of male and female intestine-infecting larvae. Exp Parasitol 37, 380–387.Google Scholar
Krupenko, DY (2014). Muscle system of Diplodiscus subclavatus (Trematoda: Paramphistomida) cercariae, pre-ovigerous, and ovigerous adults. Parasitol Res 113, 941–952.Google Scholar
La Rosa, G, Marucci, G, Rosenthal, BM & Pozio, E (2012). Development of a single larva microsatellite analysis to investigate the population structure Trichinella spiralis. Infect Genet Evol 12, 369–376.Google Scholar
Li, C, Chung, Y & Ko, R (1999). The distribution of excretory/secretory antigens during the muscle phase of Trichinella spiralis and T. pseudospiralis infections. Parasitol Res 85, 993–998.Google Scholar
Lichtenfels, JR, Murrell, D & Pilitt, PA (1983). Comparison of three subspecies of Trichinella spiralis by scanning electron microscopy. J Parasitol 69, 1131–1140.Google Scholar
Liu, ZM, Wang, C & An, CL (1991). Differentiation of the sex of Trichinella larvae collected in Changchun. Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi 9, 223–225.Google Scholar
Masetti, M, Locci, T, Cecchettini, A, Lucchesi, P, Magi, M, Malvaldi, G & Bruschi, F (2004). Nitric oxide synthase immunoreactivity in the nematode Trichinella britovi. Evidence for nitric oxide production by the parasite. Int J Parasitol 34, 715–721.Google Scholar
McCulloch, D & Gems, D (2003). Evolution of male longevity bias in nematodes. Aging Cell 2, 165–173.Google Scholar
Murata, Y, Nishiyama, T & Ishizaka, S (1998). Tomographical distribution of Trichinella spiralis (Ts) nuclei with laser scanning confocal microscopy (LSCM). Parasitol Int 47, 294.Google Scholar
Murrell, KD (2016). The dynamics of Trichinella spiralis epidemiology: Out to pasture? Vet Parasitol 231, 92–96.Google Scholar
Mutafova, T, Dimitrova, Y & Komandarev, S (1982). The karyotype of four Trichinella species. Z Parasitenkd 67, 115–120.Google Scholar
Neumann, M & Gabel, D (2002). Simple method for reduction of autofluorescence in fluorescence microscopy. J Histochem Cytochem 50, 437–439.Google Scholar
Pires-daSilva, A (2007). Evolution of the control of sexual identity in nematodes. Semin Cell Dev Biol 18, 362–370.Google Scholar
Pozio, E (2007). World distribution of Trichinella spp. infections in animals and humans. VetParasitol 149, 3–21.Google Scholar
Pozio, E, Hoberg, E, La Rosa, G & Zarlenga, DS (2009 a). Molecular taxonomy, phylogeny and biogeography of nematodes belonging to the Trichinella genus. Infect Genet Evol 9, 606–616.Google Scholar
Pozio, E & Murrell, KD (2006). Systematics and epidemiology of Trichinella. Adv Parasit 63, 367–439.Google Scholar
Pozio, E, Rinaldi, L, Marucci, G, Musella, V, Galati, F, Cringoli, G, Boireau, P & La Rosa, G (2009 b). Hosts and habitats of Trichinella spiralis and Trichinella britovi in Europe. Int J Parasitol 39, 71–79.Google Scholar
Ryzhova, O, Vus, K, Trusova, V, Kirilova, E, Kirilov, G, Gorbenko, G & Kinnunen, P (2016). Novel benzanthrone probes for membrane and protein studies. Methods Appl Fluores 4, 034007.Google Scholar
Schnell, SA, Staines, WA & Wessendorf, MW (1999). Reduction of lipofuscin-like autofluorescence in fluorescently labeled tissue. J Histochem Cytochem 47, 719–730.Google Scholar
Sednev, MV, Belov, VN & Hell, SW (2015). Fluorescent dyes with large Stokes shifts for super-resolution optical microscopy of biological objects: A review. Methods Appl Fluores 3, 042004.Google Scholar
Segliņa, Z, Bakasejevs, E, Deksne, G, Spuņģis, V & Kirjušina, M (2015). New findings of Trichinella britovi in a European beaver (Caston fiber) in Latvia. Parasitol Res 114, 3171–3173.Google Scholar
Staneva, D & Betcheva, R (2007). Synthesis and functional properties of new optical pH sensor based on benzo [de] anthracen-7-one immobilized on the viscose. Dyes Pigments 74, 148–153.Google Scholar
Staneva, D, Vasileva-Tonkova, E & Grabchev, I (2019). pH sensor potential and antimicrobial activity of a new PPA dendrimer modified with benzanthrone fluorophores in solution and on viscose fabric. J Photochem Photobiol A 375, 24–29.Google Scholar
Stewart, MT, Mousley, A, Koubkova, B, Šebelova, Š, Marks, NJ & Halton, DW (2003). Gross anatomy of the muscle systems and associated innervation of Apatemon cobitidis proterorhini metacercaria (Trematoda: Strigeidea), as visualized by confocal microscopy. Parasitology 126, 273–282.Google Scholar
Transidico, P, Bianchi, M, Capra, M, Pelicci, PG & Faretta, M (2004). From cells to tissues: Fluorescence confocal microscopy in the study of histological samples. Microsc Res Techniq 64, 89–95.Google Scholar
Villella, JB (1966). Morphologic criteria for distinguishing the sex of Trichinella spiralis larvae from muscle. J Parasitol 52, 908–910.Google Scholar
Wang, H, Liang, SC, Zhang, ZM & Zhang, HS (2004). 3-Iodoacetylaminobenzanthrone as a fluorescent derivatizing reagent for thiols in high-performance liquid chromatography. Anal Chim Acta 512, 281–286.Google Scholar
Yang, X, Liu, WH, Jin, WJ, Shen, GL & Yu, RQ (1999). DNA binding studies of a solvatochromic fluorescence probe 3-methoxybenzanthrone. Spectrochim Acta A 55A, 2719–2727.Google Scholar
Yeates, GW & Boag, B (2003). Growth and life histories in Nematoda with particular reference to environmental factors. Nematology 5, 653–664.Google Scholar
Yeates, GW & Boag, B (2006). Female size shows similar trends in all clades of the phylum Nematoda. Nematology 8, 111–127.Google Scholar
Zhytniakivska, O, Trusova, V, Gorbenko, G, Kirilova, E, Kirilov, G, Kalnina, I & Kinnunen, P (2014). Newly synthesized benzanthrone derivatives as prospective fluorescent membrane probes. J Lumin 146, 307–313.Google Scholar