Int.J.Curr.Microbiol.App.Sci (2013) 2(8): 263-271

ISSN: 2319-7706 Volume 2 Number 8 (2013) pp. 263-271 http://www.ijcmas.com

Original Research Article Encystment and encystations of invadens In-Vitro as a model to test against to the public health problem Entamoeba dispar

Ruvalcaba Ledezma Jesus Carlos1*, Latorre Cervantes Samantha2, García Romero Evelyn3, Susana Ramirez Roma4 and Juan Mora Galindo5

1Doctor of Science in Public Health, Full-time Research Professor in ICSA-UAEH, Institute of Health Sciences, Autonomous Universityof the State of Hidalgo, Pachuca Hidalgo, Mexico, academic area of Medicine and Master of Public Health. Pachuca Hidalgo, Mexico. 2Lic. In UAEH Medicine, Autonomous University of the State of Hidalgo, Pachuca Hidalgo, Mexico. 3Lic. In UAEH Medicine, Autonomous University of the State of Hidalgo, Pachuca Hidalgo, Mexico. 4Accountant CIBO- IMSS, Guadalajara Jalisco, Mexico. Science and Research 5Doctor retired CIBO-IMSS, Guadalajara Jalisco, Mexico - University of Guadalajara, Mexico *Corresponding author e-mail: [email protected]

A B S T R A C T

The life cycle of Entamoeba invadens implies the cyst formation who infest to new guests and that excystation and reproduction cause amebiasis; the excystation process is poorly K e y w o r d s understood and it hasn`t been systematically studied in vitro.The objective of this study was to quantify the excystation of cysts of E. invadens IP-1 strain, previously obtained in axenic Entamoeba conditions for incubation in medium AEM with inoculums of 2 x 105 trophozoites / ml for invadens; 72 hours. The excystation was caused to transfer the cysts to the growth medium Bl-S- cysts; 33, the first trophozoites were observed after 12 hours of incubation and after 72 hours, the trophozoites; 13% of the inoculated cyst were excystation: the most range of excystation was E. dispar; observed at 48 hours. It was obtained as cultives of trophozoites, who after six weeks it had exposue to similar growth curves to the original strain. These results demonstrated the quantitatively drugs; excystation of Entamoeba invadens and confirmed the possibility of provoke the life cycle in vitro of this in axenic conditions for facilitating the study, and it was necessary axenic to determine the optimum conditions to induce the massive and synchronize excystation. conditions; The model obtained in Entamoeba invadens IP-1 could even reduce unnecessary exposure E. histolytica to drugs, in the case of E. dispar and E. histolytica, increases the costs to the patient and costs and family, health institutions, and the risks of exposure to drugs, as E. dispar is not parasitic amoeba.

Introduction

The species pathogenic of the amoeba physical and chemical changes that the Entamoeba invadens have two stages or trophozoite does not support , the first is phases in the life cycle: the trophozoite mainly involved in the spread of the (Figure. 1), which is the form that causes parasite to new hosts (Figure. 2), this causes disease and cyst resistant to unicellular parasite of direct cycle has

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Int.J.Curr.Microbiol.App.Sci (2013) 2(8): 263-271

Figure.1 Trophozoites (t) and cysts (arrows) of Entamoeba invadens. The trophozoites present the amoeboid shape characteristic; the cysts are rounded and measured - 20 um of diameter. Differential interference contrast.

Figure.2 Growth curve of E. invadens.

264 Int.J.Curr.Microbiol.App.Sci (2013) 2(8): 263-271 an infectious stage comprising a period encystment of E. Invadens is similar in of 1 to 14 days, and their eggs can be detail to describe for E. Histolytica. transported from terrarium to terrarium Subsequently McConnachie obtained by vectors such as cockroaches and flies. Trophozoites by subculturing of cysts in the growth medium of , 2 However E. Invadens hasn't public health procedure used by others to induce the importance, as the mainly natural guests encystement (Cervantes-Mamoa and are snakes and not present safe for Martínez-Palomo, 1980; Barrer and humans, but this species of amoebae has Svihla, 1964; Thepsuparangskul et al., gained importance because of its similarity 1971). with Entamoeba histolytica (causative agent of human amebiasis) in relation with However, knowledge about the his morphology, life cycle, the cultive encystment is scarce. Knowing the factors requirements and pathogenesis, (Geiman involved in the induction about and Ratcliffe, 1936; McConnachie, 1955; encystation as the encystment, besides Diamond et al., 1978) but mainly because being an inherent basic knowledge which it is possible to obtain mature cysts of E. will introduce new strategies parasite invadens in axenic conditions, (Rengpien control by blocking their life cycle and Bailey, 1975) which to date has not avoiding the infestation of new hosts, been achieved in the case of E. histolytica. particularly of E. Histolytica contributing Thanks to this it is known the in directly relation to decreasethe ultrastructure of cysts (Chavez et al., morbidity and mortality triggers of this 1978), the ribosome s characteristics, the infestation.( Avron et al., 1982; Mora- variations in the membrane components Galindo et al., 1986). (Chayen et al., 1985) and the wall quistic composition (Arroyo-Begovich et al., The limited knowledge about encystment 1980). Similarly, we have continued to process is due to the lack of a system who study the life cycle of E. invadens by permit the massive encystment in inducing the encystment and encystment reproducibly form, because in the most in vitro. cases only has been executed qualitative analysis (Geiman and Ratcliffe, 1936; The first to study the life cycle of E. McConnachie, 1955; Cervantes-Mamoa invadens were Geiman and Radcliffe, one and Martínez-Palomo, 1980; its discoverers, (Ratcliffe and Geiman, Thepsuparangskul et al., 1971) In this 1934; Ratcliffe and Geiman, 1933), who work is evaluated quantitatively the unable to induce the massive encystment excystation of E. invadens cysts in in cultivation medium, based his studies in between Bl-S-33. observations of samples in inoculated snakes experimentally with amoebic cysts, Materials and Methods they found that are required five to seven hours for undertaking the excystation. Strain and cultivation conditions

They also observed that the emergence of This work was used trophozoites of trophozoites was affected in periods as Entamoeba invadens of the strain IP-1, short as 15 minutes or as long as 80 grown in axenic growth medium BI-S-33 minutes; they concluded that the to 25 ° C, in glass tubes with screw cap 16

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X100 mm. This strain was originally statistical evaluation we used the Student isolated from a snake (Natrix ciclopiori) in T test. 1952 and since then has been in several laboratories in different culture media. The In another series of encystment strain was maintained in our laboratory by experiments, the cultivation were washed replanting of 1x104 trophozoites / ml with 0.2% Sarkosyl for 24 hours and the every seven days. subcultivation in growth medium, to determine if the process was continued in Encystment a period of 72 hours and was determined semiquantitatively in what period occurred To Inducing the encystment were used an increased encystation; in this case was trophozoites harvested in logarithmic considered as positive the encystment growth phase, which were reseeded in observing through a microscope the tubes with seven ml of encystment presence of motile trophozoites in culture medium AEM (Rengpien, S. y G.B. tubes. Bailey, 1975), in the appropriate number for obtain 2x105 trophozoites / ml. Were Result and Discussion incubated at 25°C and to the 24, 48 and 72 hours were quantified cysts and The Cultives of E. Invadens had trophozoites in Neubauer chambers and exponential growth and yields were 3X105 we proceeded to calculated the percentage trophozoites / ml after 12 days of culture of encysted amoebae in relation to the total (Figure. 3), after which it started the cells obtained (trophozoites and cysts). declining cell period. For induction of The viability of cysts obtained at 72 hours encystment, the amoebae were harvested was determined by the exclusion of after seven to eight days of to be replanted, tripartite blue. as during this period were found in the logarithmic growth phase. Encystation As to encystment, we observed an increase The excystation of cysts was induced on in the concentration of cysts according to the same day who they were obtained, for the incubation time, the first cells in this it was incubated in growth medium encystement were of the lumps are BI-S-33 at 25 ° C after washing the normally formed in the cultives. After 48 amoebae with Sarkosyl (N-lauryl sarkosyl) hours, the average of the encystment was 0.2% to removing trophozoites. to the 50% and not significantly increased at 72 hours (Figure. 4). Were performed observations of the tubes in the inverted microscope every 12 hours up to 72 hours for detect the presence of During the process there was a trophozoites; every 24 hours were corresponding reduction in the number of quantified the trophozoites and cysts in trophozoites, those remaining as such were Neubauer chambers. For the quantitative seen primarily attached to the tubes and analysis, we calculated the percentage of presented the characteristic amoeboid trophozoites and cysts in the different form. The viability of cysts obtained was periods indicated, in relation to the total always greater to the 70%. cells in each period studied. For the

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Figure.3 Kinetic encystment of E. invadens, strain EP-1.

Figure.4 Kinetic encystment of E. invadens of the strain IP-1

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The encystement of the cysts resistant to Rengpien and Bailey (1975) were obtained sarkosyl began 12 hours after transfer the cysts from E. invadens in an AEM axenic cysts to the medium BIS-33, however, the medium, but the yield obtained was not as quantification of trophozoites in Neubauer high as reported by these authors. chambers only was possible after 24 hours (Figure. 4). No clutch is possible that our accounts were underestimated, because the cysts However, in three experiments, although obtained was agglutinated making more there were encystement determined by difficult a precise quantification. analysis inverted microscope, it was not possible to quantify the trophozoites even An alternative explanation would be that after 72 hours, the same whathappened the nutrient medium or the metabolic state between these two last. of the cells were not optimal to allow proper cell division to significantly In other seven experiments the number of increase the number of trophozoitesand in trophozoites increased in function on the direct relation was obtained a high incubation period to an average of 13%, population after of the cell lysis who and concomitantly decreased the number typically occurs in the AEM, due to their of cysts. low osmolality as compared to Bl-S-33. In previous works from our laboratory Statistical analysis showed that there (Mora-Galindo et al., 1986)15 and other significant difference between the authors, (Avron et al., 1982; Avron et al., percentage of trophozoites at 24 hours 1983) the cysts yields were also lower compared with the 48 and 72 hours, the without the authors have found the reasons same happened between the latter two. for this.

The trophozoites destruction, product of Moreover, we unknown if the trophozoites the encystement with Sarkosyl and the who wasn't encystment after 72 hours in later incubation of the cysts resistant in the medium AEM,moreover,we unknown if medium Bl-S-33, indicated who said the trophozoites who wasn't encystment process of encystement is continuous after 72 hours in medium AEM can do (Table 1), and that after 48 hours there is a incubating for prolonged periods in the higher concentration of trophozoites in same medium. relation to the 24 and 72 hours. Regarding to the encystement so far only Subsequently was subcultivated of the been performed descriptive analysis or same way that the original strain and was semiquantitative, in the latter are reported obtained a cultive with a similar growth as negative or positive, indicating with curve (Figure 3) to the strain that gave rise different number of crossings the through cysts;to differentiate it was called magnitude of the encystation. The present IP-1 strain (RL) and maintained for work report for first time quantitative several months in the laboratory, but was evaluation of the encystation. The first lost by bacterial contamination trophozoites appeared 12 hours after the (Ruvalcaba-Ledezma et al., 1989). induction of the encystation, although there is the possibility that it may occur According to what was reported by earlier. There is no difference when

268 Int.J.Curr.Microbiol.App.Sci (2013) 2(8): 263-271 compared to the time reported by Geiman previous time, may be can join the and Radcliffe(1936) although in this case amoebas that have resulted from the the encystation occurred in vivo and with a division of the trophozoites that hatched different strain of amoebae. early. Moreover, this series of experiments demonstrated that the highest encystement Not all the cysts was encystment and the percentage occurred at 48 hours. ratio of trophozoites obtained was low, possibly not all cysts were ripe, and might The obtaining of the trophozoites cultives exist anormal encystment.Although it is from cysts confirms the fact that the life generally considered that the cysts are cycle of E. invadens can be caused in resistant to sarkosyl, some might be vitro, using medium of axenic cultivation. affected by the washing with detergent. On Although in the present work it was the other hand, are unknown the causes performed a quantification of encystation, that provoke of the destruction of cysts in it was required a procedure who the the middle BI-S-33, where in theory are provoke in the synchronized form as well resistant.The destruction of cysts has as of the massive induction of the same, already been reported by other authors, for that later can be evaluated the effect of even at times not achieved trophozoites to the different factors, both on the from cysts (Chayen et al., 1985). encystement as well as on the encystation, with the goal of blocking the life cycle of It is considered that the lack of glucose these parasites and prevent infection to (Vázquez De Lara-Ceneros and Arroyo- new hosts. Begovich, 1984) and the low osmolarity in the medium (Rengpien and Bailey, 1975) Asymptomatic individuals with are the main factors that induce the documented E. histolytica infection should encystment, and it is possible that the be treated with a luminal agent to eradicate presence of glucose and osmolarity of the infection; this recommendation is based medium BI-S-33 cause the encystation of both on the known risk for the the encysted amoebae. development of invasive disease in such patients, and the fact that individual The analysis of the results corresponding shedding E histolytica cysts are a risk to in a way to the generally proposed in the public health. (Gathiram and Jackson sense that the encystement occurs in Tfhg, 1987; Haque et al., 2001) E. dispar adverse conditions and the encystation is infection does not require treatment, but performed when the conditions are own should alert the physician that the infected for developing trophozoites; however, it is person has been exposed to faecally not known which are the factors that contaminated food or water. induce the encystment, as well as at the The results of the table one explain the molecular mechanisms involved in these reason for the importance of further processes of cell differentiation. research respect to E. dispa (Samuel L Stanley, 2003) as though it is a kind of The results in Table 1 demonstrate that the amoeba comennsal represents a public encystement is a continuous process and health problem that could intervener in the trophozoites increasing Figure. 5 patients being exposed to medical results from the sum of newly hatched treatment without needing it. trophozoites more the appeared in the

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En The University of Munich between country is still used as a treatment for 2005 and 2009, 103 laboratory-confirmed amebiasis, since in the case of being amebiasis cases were detected. The mistakenly diagnosed with this disease in studycompares the results of various the case of E. dispar and E. histolytica, diagnostic tests amongthese patients, increases the costs to the patient and analyzes data on co-infections and family, health institutions, and the risks of clinicalsymptoms, and determines the risk exposure to drugs, as E. dispar is not for acquiring amebiasis.Results Initial parasitic amoeba. screening tests (stool microscopy, coproantigen enzyme-linked Acknowledgements immunosorbentassay (ELISA) were positive in 82.5 and 93.9%, respectively. The authors of the present research article Fecal samplesfrom patients with positive would like to acknowledge and truly thank screening test results were subjected to the collaboration of Yesenia Elizabeth polymerase chain reaction (PCR), which Ruvalcaba Cobián who has a B.A in detected E. histolytica in 9.7% and E. Teaching English as a Foreign Language, dispar in 88.3% of the cases. for her contributions on the revision and translation of the article; situation which The majority of E. histolytica cases and allows the possibility to increase the morethan half of the E. dispar cases had transferring and modification of scientific intestinal symptomstypical for amebiasis. knowledge. Special recognition Susana In 53.4% of the cases, intestinal Ramirez Romo and PhD Juan Mora coinfections were found, mostly Galindo for his teachings and support for Blastocystis hominis (39.8%), Giardia this project lamblia (10.7%), Campylobacter spp. (4.9%), and Salmonella typhi (2.9%). The The authors declare that no conflict of risk for travelersto be infected with E. interests for the publication of this histolytica or E. dispar was highestfor research paper. destinations in West Africa, East Africa, and South and South-East Asia. References (Herbinger et al., 2011) In general terms it is necessary to continue investigating the Arroyo-Begovich, A., A. Carabez-Trejo and matter and that is why we consider it Ruiz-Herrera, J. 1980. Identification of the important to review this article again. structural component in the cyst wall of Entamoeba invadens. J. Parasitol 66:735. Using the methodology was possible to Avron, B., R. Bracha, M.R. Dustsch and cultivate, encysting Entamoeba invadens Mirelman, D. 1983. Entamoeba invadens and desenquistar in axenic in vitro. The and Entamoeba histolytica: Separation cysts and trophozoites obtained both in- and purification of precysts and cysts by vitro use of Entamoeba invadens allows centrifugation on discontinuous density amoeba that species as a model to test the gradients of percoll. Exp.Parasitol. 55:265. effect of drugs amebicides as E. invadens Avron, B., R.N., Deutsch and Mirelman, D. represents a model for studies of amebiasis 1982. Chitin synthesis inhibitors prevent cyst formation by Entamoeba invadens. caused by E. histolytica. The model Biochem. Biophys. Res. Comum. obtained in Entamoeba invadens IP-1 108:815. could even reduce unnecessary exposure to drugs such as in our

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