In Vitro Culture of Neoechinorhynchus Buttnerae
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Original Article ISSN 1984-2961 (Electronic) www.cbpv.org.br/rbpv Braz. J. Vet. Parasitol., Jaboticabal, v. 27, n. 4, p. 562-569, oct.-dec. 2018 Doi: https://doi.org/10.1590/S1984-296120180079 In vitro culture of Neoechinorhynchus buttnerae (Acanthocephala: Neoechinorhynchidae): Influence of temperature and culture media Cultivo in vitro de Neoechinorhynchus buttnerae (Acanthocephala: Neoechinorhynchidae): influência da temperatura e dos meios de cultura Carinne Moreira de Souza Costa1; Talissa Beatriz Costa Lima1; Matheus Gomes da Cruz1; Daniela Volcan Almeida1; Maurício Laterça Martins2; Gabriela Tomas Jerônimo2* 1 Programa de Pós-graduação em Aquicultura, Universidade Nilton Lins, Manaus, AM, Brasil 2 Laboratório Sanidade de Organismos Aquáticos – AQUOS, Departamento de Aquicultura, Universidade Federal de Santa Catarina – UFSC, Florianópolis, SC, Brasil Received August 9, 2018 Accepted September 10, 2018 Abstract Infection by the acantocephalan Neoechinorhynchus buttnerae is considered one of most important concerns for tambaqui fish (Colossoma macropomum) production. Treatment strategies have been the focus of several in vivo studies; however, few studies have been undertaken on in vitro protocols for parasite maintenance. The aim of the present study was to develop the best in vitro culture condition for N. buttnerae to ensure its survival and adaptation out of the host to allow for the testing of substances to be used to control the parasite. To achieve this, parasites were collected from naturally infected fish and distributed in 6-well culture plates under the following treatments in triplicate: 0.9% NaCl, sterile tank water, L-15 Leibovitz culture medium, L-15 Leibovitz + agar 2% culture medium, RPMI 1640 culture medium, and RPMI 1640 + agar 2% culture medium. The plates containing the parasites were maintained at 24 °C, 28 °C, and 32 °C. The RPMI 1640 + agar 2% culture medium showed the best survival of 24 days at 24 °C. No body alterations such as swollen parasites, body deformation, dehydration and hardening were observed in the RPMI 1640 + 2% culture medium. Keywords: Fish farming, tambaqui, acantocephalan, culture, survival. Resumo A infecção pelo acantocéfalo Neoechinorhynchus buttnerae é considerada uma das preocupações mais importantes para produção de tambaqui (Colossoma macropomum). Estratégias de tratamento têm sido o foco de vários estudos in vivo; entretanto, poucos estudos foram realizados em protocolos in vitro para manutenção do parasito. O objetivo deste estudo foi desenvolver a melhor condição de cultura in vitro para N. buttnerae para garantir sua sobrevivência e adaptação fora do hospedeiro, a ser utilizado para teste com substâncias no controle do parasito. Para isso, os parasitos foram coletados de peixes naturalmente infectados e distribuídos em placas de cultura de 6 poços sob os seguintes tratamentos em triplicata: 0.9% NaCl, água estéril do tanque, meio de cultura L-15 Leibovitz, meio de cultura L-15 Leibovitz + ágar 2%, meio de cultura RPMI 1640, e meio de cultura RPMI 1640 + ágar 2%. As placas contendo os parasitos foram mantidos a 24 °C, 28 °C, e 32 °C. O meio de cultura RPMI 1640 + ágar 2% apresentou a melhor sobrevivência de 24 dias a 24 °C. Nenhuma alteração corporal tais como inchaço dos parasitos, deformação corporal, desidratação e endurecimento foram observados no meio de cultura RPMI 1640 + ágar 2%. Palavras-chave: Piscicultura, tambaqui, acantocéfalo, cultivo, sobrevivência. Introduction Intensive fish farming may provoke environmental alterations a decrease in fish immune systems, which enhance susceptibility to that favor disease outbreaks (REVERTER et al., 2014). Changes in infections (QUESADA et al., 2013). Among the main pathogens temperature, water quality, and nutritional status are responsible for causing diseases in fish farms are protozoans, myxozoans, helminths, and crustaceans, which under favorable environmental conditions may reproduce or disseminate culminating in diseases *Corresponding author: Gabriela Tomas Jerônimo. Laboratório Sanidade (JERÔNIMO et al., 2015). de Organismos Aquáticos – AQUOS, Departamento de Aquicultura, Universidade Federal de Santa Catarina – UFSC, Rod. Admar Gonzaga, 1346, The acanthocephalanNeoechinorhynchus buttnerae (Golvan, 1956) CEP 88040-900, Florianópolis, SC, Brasil. e-mail: [email protected] belongs to the class Eoacanthocephala (Van Cleve, 1936), order This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. v. 27, n. 4, oct.-dec. 2018 In vitro culture of Neoechinorhynchus buttnerae 563/569 563 Neoechinorhynchida (Ward, 1917), and family Neoechinorhynchidae Previous studies have not evaluated the influence of temperature (Travassos, 1917), and is an endoparasite mostly found in the on parasite survival. Nevertheless, a study by Thomas (2002) stated intestines of their hosts (SANTOS et al., 2013). Its infection that natural environment seasonal variations, e.g., water temperature has been considered one of the most important obstacles for changes, might influence the abundance of helminth parasites. tambaqui (Colossoma macropomum Cuvier 1816) production in The aim of the present study was to evaluate the best Brazil (LOURENÇO et al., 2017) and can cause economic losses culture medium and temperature for the in vitro culture of the (GOMES et al., 2017). This is related to a significant decrease in acanthocephalan N. buttnerae, an important parasite of tambaqui fish production and fish performance owing to weight loss, cachexia C. macropomum in the Amazon. due to competition for the diet nutrients, as well as decreased area of intestinal absorption (JERÔNIMO et al., 2017). Moreover, the parasite attaches to the intestinal epithelium with their hooks, Material and Methods induces a severe inflammatory reaction, and causes dilacerations of the intestinal mucosa (JERÔNIMO et al., 2017). Parasites and experimental design Acanthocephalosis in Brazil represents an important concern due to high infections of fish in the northern region (MALTA et al., 2001; A total of 13 fish (19.15 ± 2.30 cm; 138.81 ± 52.57 g) naturally GOMES et al., 2017; LOURENÇO et al., 2017; MATOS et al., parasitized with the acanthocephalan N. buttnerae were used to 2017). Therefore, the development of effective treatments against obtain the parasites after dissection. After the removal of parasites, the parasite is urgently required. Prior to in vivo treatment, the standardization of techniques in vitro using parasites out of their they were washed in 0.9% NaCl for posterior distribution in 6-well hosts is strongly recommended. Both studies are required because of plates containing 5 mL of each tested solution, with 5 parasites per the metabolic dependence of the parasite in the microenvironment well. This procedure was performed under a laminar flow cabinet. (POULIN & MORAND, 2004). In the past, studies have tested The experimental design was completely randomized and was the acanthocephalan culture in vitro; however, the methods have composed of six treatments with three replicates for each treatment. not been well-defined (NICHOLAS & GRIGG, 1965) and, when These included parasites maintained in 0.9% NaCl, sterile tank successful, the parasites were kept alive only for a short period water, L-15 (Leibovitz) culture medium, L-15 (Leibovitz) + agar (POLZER & TARASCHEWSKI, 1994). 2% culture medium, RPMI 1640 culture medium, and RPMI The first trial of in vitro culture was undertaken by Gettier 1640 + agar 2% culture medium. The plates were incubated in (1942) with 25 days survival of N. emydis (Leidy, 1852) which is a a B.O.D (model TE 391) at 24 °C, 28 °C, and 32 °C based on parasite of turtles, in 0.5% NaCl added to 0.02% CaCl In a later 2. the procedure described in Oliveira & Val (2017) taking into study, Van Cleave & Ross (1944) reached 13 days survival of this consideration that tambaqui inhabits waters of 25 °C to 34 °C parasite in 0.7% NaCl. The ideal culture medium for the growth mean temperature. of Angiostrongylus cantonensis (Chen, 1935) larvae was found to be a CBSS-based solution containing 10% L-15 (Leibovitz) (Gibco) Parasites were observed at 24 h intervals because a study by Van culture medium, 10% tryptose phosphate broth (Gibco), 20% Cleave & Ross (1944) showed that more frequent observations fetal calf serum, and 26 mM of sodium bicarbonate. Fifty days of parasites are responsible for reduced survival times. During after culture, 82% of the larvae had changed to the third stage incubation, the parasites were evaluated on their movement after (HATA & KOJIMA, 1990). Thus, L-15 (Leibovitz) medium gently stimulus with a needle and their morphological characteristics began to be used for growth and maintenance of cell tissue for were noted. Parasites were considered dead when no movements virus diagnostics (LEIBOVITZ, 1963). As previously observed, were seen after stimulus was applied. in vitro studies with L-15 (Leibovitz) medium have demonstrated efficacy for culturing parasites (SCHRAMLOVÁ et al., 1984; HATA Culture medium preparation and description & KOJIMA, 1990). Recently, this technique was improved by Buron et al. (2009) who studied the culture of acanthocephalans Moniliformis moniliformis (Bremser, 1811) Travassos, 1915, in rats L-15 (Leibovitz) for 8 days in medium enriched with glucose. Conversely, Roswell Park Memorial Institute (RPMI) 1640 The L-15 (Leibovitz)