In vitro Culture and Morphology of Fish Trypanosomes from South American Wetland Areas

In vitro Culture and Morphology of Fish Trypanosomes from South American Wetland Areas Alyssa Rossi Borges1*, Moara Lemos2, Drausio Honorio Morais3, Thaïs Souto-Padrón4 and Marta D’Agosto5 1Universidade Federal de Juiz de Fora, Colégio de Aplicação João XXIII, Departamento de Ciências Naturais, Rua Visconde de Mauá 300, Santa Helena, 36015260, Juiz de Fora, MG, Brazil 2Institut Pasteur, Department of Trypanosoma Cell Biology, Rue Dr Roux 25-28, 75015, Paris, France 3Universidade Regional do Cariri, Centro de Ciências Biológicas e da Saúde, Departamento de Ciências Biológicas, Campus do Pimenta, Rua Cel. Antonio Luiz, 1161, Bairro do Pimenta, 63105-100, Crato, CE, Brazil 4Universidade Federal do Rio de Janeiro, Instituto de Microbiologia Professor Paulo de Góes, Laboratório de Biologia Celular e Ultraestrutura, Rio de Janeiro, RJ, Brazil 5Universidade Federal de Juiz de Fora, Instituto de Ciências Biológicas, Departamento de Zoologia, Laboratório de Protozoologia, Rua José Lourenço Kelmer, s/n, Bairro São Pedro, Juiz de Fora, 36036-900, MG, Brazil SOJ Microbiology & Infectious Diseases Open Access Research Article

have been identified in freshwater and marine fish worldwide usually based on morphology of blood trypomastigotes and the hypothesis of parasite-host specificity [2][3][4][5][6][7].However, certain fish trypanosome species are pleomorphic and are not specific to the vertebrate host [8][9][10].Thus, the access to molecular data information is growing in an attempt to supplement current morphological and morphometric data, and to clarify the species identification [9,[11][12][13].
Although the characterization and taxonomy of fish trypanosomes has been improved through the use of molecular approaches [8,[11][12][13], mixed infections that normally occur in nature can limit its accuracy [9,14].Therefore, the achievement of in vitro isolates and the establishment of laboratory clonal lineages are becoming increasingly important [9,14].Besides this application, culturing can provide a great number of parasites that can be used in different studies, providing important information regarding their biology and ultrastructural features [9], in addition to the antigenic characteristics of parasites and their relationship with the host immune system [15,16].
In fact, the pathogenic effects of fish trypanosomes species are not completely known.Some experimental infections studies have demonstrated that Trypanosoma danilewskyi (synonym Trypanosoma carassii) infections can lead the host to develop anemia and anorexia [17,18].In addition, the investigation of natural infections with other trypanosomes reveal their potential role as pathogen inducing alterations of hematological parameters [19,20] and on total body weight of fish [21].However, other investigations suggested there is a delicate balance between fish trypanosomes and host immune system, which can lead to the persistence of the parasite in low intensity and host survival [22].

Abstract
Fish trypanosomes are a group of parasites with taxonomic issues and can demonstrate potential pathogenic effects to the host.Nowadays, producing in vitro isolates of fish trypanosomes has become a key point to better understand their biology and taxonomy.Despite of that fact, to date only one species of fish trypanosome was isolated in South America.In addition, there is little information about trypanosomes that occur in Pantanal, an important World Conservation Complex.The purpose of this study was to investigate trypanosome infections in fish from hyperseasonal Savanna in the Brazilian Pantanal wetlands by producing in vitro isolates.During the dry period, 74 fish representing 4 species were collected from 5 lagoons, and blood was sampled by cardiac or caudal vein puncture.Analysis of blood smears and in vitro cultures showed that 7.31% of hosts were infected by trypanosomes.The in vitro isolation and maintenance were made by using Ponselle blood agar base without NaCl and 60% Eagle Basal Medium and Novy-MacNeal-Nicolle medium mixed with fish Ringer's solution.Here, we have highlighted the importance of combining different diagnostic techniques to adequately identify trypanosome infections in fish and have provided a brief morphological description and morphometric features of 20 blood trypomastigotes.In addition, this is the first report of trypanosomes isolated from fish of the Pantanal wetlands, which can contribute to future studies of ultrastructural characteristics, hostparasite interactions, or molecular biology.

Introduction
The first record of a trypanosome was likely made in 1841, when parasites were observed in the blood of Salmo fario [1].Since this observation, more than 200 trypanosome species Special Issue Title: Trypanosomatids Biology In Brazil, studies on fish trypanosomes have focused on their occurrence and the identification of species.Traditionally, species identification has been based upon morphological features of blood trypomastigotes, along with the host specificity hypothesis [4,6,23,24].At least 64 nominal species of fish trypanosomes have been recorded to date, with most of them identified using morphological parameters exclusively [3,9].Only recently, trypanosomes from armored catfish were isolated and maintained in vitro using 9 different culture media and one new nominal species could be identified as Trypanosoma abeli [9,26].Altogether, those data shown the scarcity of knowledge regarding fish trypanosomes species that really occur in Brazil [9] and their role as pathogens of fish.Therefore, cultivating in vitro isolates represents a key point in studies of those parasites, which can allow new studies regarding their molecular taxonomy as well as other investigations under controlled laboratory conditions, such as their role as pathogens of fish.
The hyperseasonal Savanna of the Brazilian Pantanal wetlands is an important ecosystem in the Brazilian Midwest that is considered a World Heritage of Humanity, and it contains more than 260 species of fish [27].The present knowledge of the various trypanosomes that occur in these fish species is limited to infections described in two fish species: Trypanosoma sp. in Gymnotus aff.inaequilabiatus [28] and Trypanosoma azoubeli in Pterodoras granulosus [29].In our study, we investigated trypanosome infections in fish of the Pantanal wetlands.We examined the morphological features of trypomastigotes in blood and conducted in vitro isolation of epimastigotes and trypomastigotes.

Fish collection and study area
Seventy-four fish were collected during the dry period and were identified as Gymnotus sp.Fish were anaesthetized using eugenol diluted in water (50 mg/ L) [26] and were examined for the presence of leeches.Blood samples were taken by cardiac or caudal vein puncture following the recommendations of the Ethics Committee for Animal Experimentation (protocol number 025/ 2011) of the Federal University of Juiz de Fora (MG, Brazil).

Prevalence and intensity of infection
Blood smears were prepared using 20 µL of blood, stained with 9% Giemsa solution, and screened by light microscopy (1,000× magnification) to detect trypanosomes.To estimate the intensity of infection (expressed in parasites/ mL), all parasites found in 1 cm 2 of the smear (250 microscopic fields in 1,000× magnification) were recorded and calculated as follows: 12 cm 2 -----20 µL of blood n° of parasites ----0.75µL 1 cm 2 -----0.75µL of blood x ----1000 µL "n° of parasites" represents the number recorded in 1 cm 2 of smears "x" represents the estimated number of parasites in 1 mL of blood The overall prevalence of trypanosomes was calculated as described previously [30] by using blood smears analysis and in vitro culture isolation.

Morphology and morphometry of blood trypomastigotes
Parasites were photographed and measured using Image-Pro Plus ® 5.0 (Media Cybernetics, Rockville, MD, USA).The measurements recorded included total body length with flagellum (TL), body length along the cell midline (BL), body width at the center of the nucleus (BW), length of the free flagellum (F), Nucleus Length (NL), nucleus width at the center of the nucleus (NW), distance from the center of the nucleus to the anterior of the cell (NA), distance from the center of the nucleus to the posterior of the cell (NP), distance from the center of the kinetoplast to the center of the nucleus (KN), Kinetoplast Length (KL), Kinetoplast Width (KW), and distance from the center of the kinetoplast to the posterior of the cell (KP).The nuclear index (NI = NP/ NA) and kinetoplast index (KI = NP/ KN) were also calculated (Figure 1).

In vitro cultivation of trypanosomes
The trypanosomes were isolated using three biphasic culture media: Ponselle blood agar base without NaCl (PO), mixed with 60% Eagle Basal Medium (BME) and supplemented with 20 µg/ mL hemin and 10% heat-inactivated Fetal Calf Serum (FCS); Blood Agar Base (BAB) and 50% BME, supplemented with 20 µg/ mL hemin and 10% heat-inactivated FCS; and Novy-MacNeal-Nicolle (NNN) medium mixed with fish Ringer's solution as described previously [26].Following inoculation with blood samples, cultures were kept in room temperature for 5 days and then transferred to a BOD incubator (Eletrolab) at 22°C.For positive cultures, trypanosomes were transferred to new tubes containing the same medium every 7 days.

Prevalence and intensity of infection
The total prevalence of trypanosome infections in Pterygoplichthys sp. was 7.31% (3/ 41) and one of these had the infection detected only by in vitro isolation.Analysis of blood smears showed that Pterygoplichthys sp. was infected with trypanosomes, with an intensity of 9 × 10 -4 parasites/ mL Blood smear analysis and culture isolation failed to reveal the presence of trypanosomes in any other fish species.In addition, all fish were inspected for the presence of leeches, but none were found.

Morphological aspects of blood trypomastigotes
The trypanosomes observed showed deeply Giemsa staining of the cytoplasm with some small vacuoles along the body.The nucleus was oval and displaced toward to anterior region of the body (NI > 1).The kinetoplast was circular or oval and located at the posterior extremity of the body (KI < 2).The flagellum emerged from the posterior end of the body and followed the outline causing ripples, finishing in a long free portion (Figure 2A-C).Some of the parasites had striations along their body length (Figure 2C).The morphometric analysis results for 20 trypomastigotes are shown in Table 1.

In vitro cultivation and maintenance of trypanosomes
Following the inoculation of PO/ 60% BME and NNN/ fish Ringer's solution cultures with trypomastigotes from blood, we observed epimastigotes (Figure 2D) and trypomastigotes (Figure 2E) in vitro.The period required for trypomastigotes to differentiate into epimastigotes could not be recorded in this study because of the field conditions.In vitro epimastigotes had an elongated body with a round kinetoplast (Figure 2D), whereas trypomastigotes had a slender and shorter body when compared with the blood forms (Figure 2E).Trypomastigotes and epimastigotes divided by binary fission (not shown) and were usually clustered with the anterior or posterior extremities in the center (not shown).

Discussion
We have demonstrated a low prevalence of trypanosome infection among Pterygoplichthys sp.This genus of fish belongs to the Loricariidae family and is representative of the hosts with the greatest number of trypanosome infections in Brazil.It was previously shown that the prevalence of trypanosomes in Hypostomus punctatus was 100% [32], whereas trypanosome prevalence ranged from 22.6-100% in 6 other species of armored catfish [19].These differences in prevalence among fish species could be related to the behavior of the host [19], the abundance of the vector in the environment [5,33], and the diagnostic method used [28,32].
It has been confirmed that the examination of fresh blood is more sensitive than the microhematocrit method and blood smears [28,32].Although recently a prevalence of 100% has been recorded in armored catfish through analysis of blood smears

In vitro Culture and Morphology of Fish Trypanosomes from South American Wetland Areas
Copyright: © 2016 Borges et al. [9], this technique is considered of low sensitivity.Polymerase Chain Reaction (PCR) assays represents other efficient method for fish trypanosomes diagnose [8].However, PCR amplification of the trypanosomes DNA were not achieved for the positive hosts in this study, which can be related to the low intensity of trypanosomes in total blood or the quality of the material stored.In fact, the sensitivity of the various diagnostic methods available for use could be affected by the intensity of the infection, which is known to vary according to the stage of infection [18,34].Indeed, we used the same blood smear screening techniques described in a previous study [9], but the prevalence and intensity were much lower (9 × 10 -4 parasites/ mL in this study compared with 1 × 10 2 parasites/ mL [9]).
In vitro isolation of fish trypanosomes is laborious and timeconsuming, especially when the field conditions for primary isolations are not propitious, such as the lack of laboratory structure, the geographical isolation of collection sites and the high temperatures exhibited.Furthermore, various media components, temperature, and pH can affect the growth of the parasites [35,36].Initial attempts to isolate fish trypanosomes in Brazil were unsuccessful [37,38]; however, T. abeli was recently isolated from armored catfish [26].We were able to isolate trypanosomes from fish of the Brazilian Pantanal wetlands for the first time, providing new information regarding the diversity of fish trypanosomes and enabling future studies of their biology.The biphasic media used were found to be more effective than monophasic media with respect to the maintenance of fish trypanosomes [26].Previous findings suggest that diffusion of nutrients from the blood agar base is necessary for the cultivation of trypanosomes [39].The first trypomastigotes observed in culture resemble the blood forms and were replaced by epimastigotes, and then shorter trypomastigotes grew, which have been previously observed by other groups [21,26].
In the present study, we have provided a brief morphological description and presented morphometric features of trypanosomes from Pterygoplichthys sp.We also successfully isolated trypanosomes from Pantanal fish for the first time and demonstrated the importance of combining diagnostic techniques for the identification of trypanosome infections.The low number of parasites found in blood and the lack of molecular data prevented the morphotype classification; however, the present findings will lay an impact on future studies on Pantanal fish trypanosomes, showing that is possible to achieve their culturing besides the field adversities, which can promote other investigations regarding not only their taxonomy as well as their ultrastructural features and metabolic requirements.

Figure 1 :
Figure 1: Morphological parameters measured in blood trypomastigotes for morphological characterization [adapted from 31].Total Body Length With Flagellum (TL), Body Length Along the Cell Midline (BL), Body Width at the Center of the Nucleus (BW), length of the free flagellum (F), Nucleus Length (NL), Nucleus Width at the Center of the Nucleus (NW), Distance from the center of the nucleus to the anterior of the cell (NA), Distance from the center of the nucleus to the posterior of the cell (NP), Distance from the center of the kinetoplast to the center of the nucleus (KN), Kinetoplast Length (KL), Kinetoplast Width (KW), and Distance from the center of the kinetoplast to the posterior of the cell (KP).

Figure 2 :
Figure 2: Morphological features of Trypanosoma sp. from Pantanal wetland fish.(A, B) Elongated trypomastigotes with nucleus (N), Kinetoplast (k), and Flagellum (F) indicated.(C) Trypomastigote with striations (S) along the body, and a short flagellum.(D) Various epimastigote forms observed in cultures.(E) Short and slender trypomastigote with a rod-like kinetoplast observed in Ponselle blood agar base without NaCl mixed with 60% Eagle Basal Medium.The scale bars (A-E) indicate 10 µm.

Table 1 :
Morphometric features of Trypanosoma sp. in the blood of Pterygoplichthys sp. from Brazilian Pantanal.The measures are expressed in µm.
* The codes are presented in the methods section