Simple and Rapid Staining for Detection of Entamoeba Cysts and Other Protozoans With Fluorochromes

Kawamoto, Fumihiko; Mizuno, S.; Fujioka, Hisashi; Kumada, Nobuo; Sugiyama, Etsuro; Takeuchi, Tsutomu; Kobayashi, Seiki; Iseki, Motohiro; Yamada, Minoru; Matsumoto, Yoshitsugu; Tegoshi, Tatsuya; Yoshida, Yukio

doi:10.7883/yoken1952.40.35

Cited by 16 publications

(2 citation statements)

References 11 publications

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“…In the current study, we have characterized the cyst-like structure using two fluorescent dyes that uniquely bind to chitin: Calcofluor White ( 74 ) and Alexafluor-conjugated Wheat Germ Agglutinin, or WGA ( 75 ). We have demonstrated that T. foetus cyst-like structures, in contrast to trophozoites, were resistant to detergent and did not allow the diffusion of Evans blue into the interior of the cell.…”

Section: Discussionmentioning

confidence: 99%

Prolonged survival of venereal Tritrichomonas foetus parasite in the gastrointestinal tract, bovine fecal extract, and water

Martínez,

Iriarte,

Salas

et al. 2023

Microbiol Spectr

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Bovine tritrichomonosis is a venereal disease that causes economic losses around the world. Over the last 100 years, the life cycle of the protozoan Tritrichomonas foetus in bovines has only considered venereal transmission, almost exclusively through natural mating from an infected animal to a healthy animal. Here, we provide the first direct evidence that T. foetus can survive the passage through the gastrointestinal tract in bovines. We demonstrated that the parasite can be discharged by feces in orally infected animals and contaminate the cow’s reproductive tract. We showed that the parasite is capable of surviving at pH conditions usually found along the bovine gastrointestinal tract, and our findings suggest that the parasites could survive in bovine feces and water for several days, probably through the formation of resistant pseudocysts or cyst-like structures. Taken together, our results demonstrate that T. foetus is more resistant to adverse external conditions than previously considered, which could be relevant for the life cycle of this parasite in bovines. IMPORTANCE Nowadays, the routine herd diagnosis is usually performed exclusively on bulls, as they remain permanently infected, and prevention and control of Tritrichomonas foetus transmission are based on identifying infected animals and culling practices. The existence of other forms of transmission and the possible role of pseudocysts or cyst-like structures as resistant forms requires rethinking the current management and control of this parasitic disease in the future in some livestock regions of the world.

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Section: Discussionmentioning

confidence: 99%

Prolonged survival of venereal Tritrichomonas foetus parasite in the gastrointestinal tract, bovine fecal extract, and water

Martínez,

Iriarte,

Salas

et al. 2023

Microbiol Spectr

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“…Presence of chitin is a characteristic feature of cysts in other protozoan parasites like Entamoeba histolytica and Giardia lamblia (Arroyo-Begovich et al, 1980;Ward et al, 1985). Thus, using two fluorescent dyes which uniquely bind to chitin [Calcofluor White; CFW (Kawamoto et al, 1987) and Alexafluor-conjugated Wheat Germ Agglutinin; WGA (Chatterjee et al, 2009)], we have scored for encystation and excystation in axenic in vitro culture of lab passaged as well as fresh human clinical isolates of T. vaginalis. Thus, without the use of any temperature perturbations and/or chemical agents, we were successful in achieving encystation and excystation of T. vaginalis in vitro.…”

Section: Introductionmentioning

confidence: 99%

Demonstration and Characterization of Cyst-Like Structures in the Life Cycle of Trichomonas vaginalis

Beri

Yadav

Devi

et al. 2020

Front. Cell. Infect. Microbiol.

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Trichomonas vaginalis is the parasitic protozoan residing in human urogenital tract causing trichomoniasis, which is the leading non-viral sexually transmitted disease. It has cosmopolitan distribution throughout the globe and affects both men and women. Lifecycle of the parasite has been traditionally described as consisting of motile and symptom-causing trophozoites. Chemical and temperature perturbations in trophozoites have been shown to aid conversion to pseudocysts, which is poorly investigated. In the current study, we show the formation of viable cyst-like structures (CLS) in stationary phase of T. vaginalis axenic culture. We used a fluorescent stain called calcofluor white, which specifically binds to chitin and cellulose-containing structures, to score for T. vaginalis CLS. Using flow cytometry, we demonstrated and quantitated the processes of encystation as well as excystation; thus, completing the parasite's lifecycle in vitro without any chemical/temperature alterations. Like cysts from other protozoan parasites such as Entamoeba histolytica and Giardia lamblia, T. vaginalis CLS appeared spherical, immotile, and resistant to osmotic lysis and detergent treatments. Ultrastructure of CLS demonstrated by Transmission Electron Microscopy showed a thick electron-dense deposition along its outer membrane. To probe the physiological role of CLS, we exposed parasites to vaginal pH and observed that trophozoites took this as a cue to convert to CLS. Further, upon co-culturing with cells of cervical origin, CLS rapidly excysted to form trophozoites which abrogated the cervical cell monolayer in a dose-dependent manner. To further corroborate the presence of two distinct forms in T. vaginalis, we performed two-dimensional gel electrophoresis and global, untargeted mass spectrometry to highlight differences in the proteome with trophozoites. Interestingly, CLS remained viable in chlorinated swimming pool water implicating the possibility of its role as environmentally resistant structures involved in non-sexual mode of parasite transmission. Finally, we showed that symptomatic human patient vaginal swabs had both T. vaginalis trophozoites and CLS; thus, highlighting its importance in clinical infections. Overall, our study highlights the plasticity of the pathogen and its rapid adaption when subjected to stressful environmental cues and suggests an important role of CLS in the parasite's life cycle, pathogenesis and transmission.

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Parasites of Rats and Mice

Baker¹

2007

Flynn's Parasites of Laboratory Animals

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Simple and Rapid Staining for Detection of Entamoeba Cysts and Other Protozoans With Fluorochromes

Cited by 16 publications

References 11 publications

Prolonged survival of venereal Tritrichomonas foetus parasite in the gastrointestinal tract, bovine fecal extract, and water

Prolonged survival of venereal Tritrichomonas foetus parasite in the gastrointestinal tract, bovine fecal extract, and water

Demonstration and Characterization of Cyst-Like Structures in the Life Cycle of Trichomonas vaginalis

Parasites of Rats and Mice

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