Interplay between Histomonas meleagridis and Bacteria: Mutualistic or Predator-Prey?

Histomonas meleagridis is an extracellular protozoan parasite and the aetiological agent of histomonosis, an important poultry disease whose impact is greatly accentuated by inaccessibility of any treatment. A special feature of the parasite is its intricate interplay with bacteria in vitro and in vivo, the focus of this article.

Escherichia coli strongly supports the growth of Histomonas meleagridis, in a monoxenic culture, without influence on its pathogenicity.

Based on clonal cultures of Histomonas meleagridis, monoxenic cultures have, to our knowledge for the first time, been established in a liquid medium. The faecal flora was exchanged for defined bacterial strains by selective destruction of the initial bacteria with a variety of antibiotics, keeping the flagellate alive. The growth of the protozoan parasite was found to depend on the bacteria, especially on their energy metabolism. Escherichia coli was found to strongly support the growth of the parasite, whereas Salmonella enterica serovar Typhimurium and Pseudomonas aeruginosa were less efficient. Confocal laser microscopy showed that H. meleagridis could take up green fluorescent protein-tagged E. coli DH5α, suggesting that bacteria serve as a food supply for the protozoa. By exchanging the bacterial flora for E. coli strain DH5α in H. meleagridis cultures that underwent continuous in vitro passages, it was possible to show that the in vivo attenuation process was independent of the bacteria. Furthermore, the gut flora in infected turkeys had no negative effect on the protozoan's virulence. Consequently, attenuation depends not on the bacteria in the culture but on the in vitro passages. Finally, the experiments provided evidence that the infection of turkeys with H. meleagridis enabled infection of the liver with E. coli.

Axenization and optimization of in vitro growth of clonal cultures of Tetratrichomonas gallinarum and Trichomonas gallinae.

A rapid and simple procedure was established to obtain clonal axenic cultures of Tetratrichomonas gallinarum and Trichomonas gallinae and to optimize their in vitro growth conditions. Medium 199 was used for axenization of two genetically different clones of T. gallinarum and T. gallinae. Six different media were used to optimize the growth behaviour of axenically grown parasites: Medium 199, TYM, TYI-S-33, Hollander fluid (HF), Trichomonas vaginalis (TV) and modified TV media. The highest cell yields for both axenic clones of T. gallinarum were obtained in modified TV medium without antibiotics. The maximum numbers of trophozoites of T. gallinae were obtained in an optimized HF medium. This study demonstrated that axenic cultures for T. gallinarum and T. gallinae could be obtained avoiding the migration technique through a V-tube. Following axenization and optimization, both clones of T. gallinarum and T. gallinae could be propagated both aerobically and anaerobically.

Ectobiotic spirochetes of flagellates from the termite Mastotermes darwiniensis: attachment and cyst formation.

The association of the gut flagellates Mixotricha paradoxa and Deltotrichonympha sp. from the termite Mastotermes darwiniensis with ectobiotic spirochetes and bacterial rods is investigated with light and electron microscopy. Treatment with different chemicals disturbing molecular interactions and use of the freeze-fracture and freeze-etch technique show that hydrophobic interactions and integral membrane proteins seem to be involved in the firm attachment at the contact sites. Application of antibiotics reduces the number of ectobionts and leads to a disintegration of the cortical attachment systems. As a result Mixotricha becomes spherical and immotile. In both flagellates the antibiotics have a further effect: they lead to a transformation of some of the spirochetes into cystic bodies. Cyst formation of ectobiotic spirochetes is here reported for the first time. Starvation has a similar but less dramatic influence than antibiotics. The cysts contain protoplasmic cylinders in the periphery and sometimes larger central bodies. Production of dormant cystic forms may be a survival mechanism under hostile conditions.

Devescovinid features, a remarkable surface cytoskeleton, and epibiotic bacteria revisited in Mixotricha paradoxa, a parabasalid flagellate.

This work reports on the flagellate systematics and phylogeny, cytoskeleton, prokaryote-eukaryote cell junction organisation, and epibiotic bacteria identification. It confirms the pioneer 1964 study on Mixotricha paradoxa and supplies new information. Mixotricha paradoxa has a cresta structure specific to devescovinid parabasalid flagellates, a slightly modified recurrent flagellum, and an axostylar tube containing two lamina-shaped parabasal fibres. However, many parabasal profiles are distributed throughout the cell body. There is a conspicuous cortical microfibrillar network whose strands are related to cell junction structures subjacent to epibiotic bacteria. The supposed actin composition of this network could not be demonstrated with anti-actin antibodies or phalloidin labelling. Four types of epibiotic bacteria were described. Bacillus-shaped bacteria with a Gram-negative organisation are nested in alternate rows on most of the surface of the protozoon. They induce a striated calyxlike junction structure beneath the adhesion zone linked to the cortical microfibrillar network. Slender spirochetes are attached by one differentiated end to the plasma membrane of the protozoon, forming knobs on the cell surface. Two very similar long rod-shaped bacteria are also attached on the knobs of the plasma membrane. A large spirochete attributed to the genus Canaleparolina is also attached to the protozoon. Observations on epibiotic bacteria and of their attachments are compared with several described epibiotic bacteria of symbiotic protozoa and with the results of the molecular identification of the epibiotic bacteria of M. paradoxa.

Electron microscopic study of Tetratrichomonas didelphidis and its interaction with a prokaryotic cell.

Tetratrichomonas didelphidis is a flagellate protozoan found in the intestine of the opossum. The parasite lives in a hostile and stressed environment, where it interacts with microorganisms and can survive under extreme conditions for growth, involving strict anaerobiosis or equilibration with air and abundance or absence of nutrients. The in vitro cultivation of this protozoan depends upon Escherichia coli as a growth-promoting partner. In this study, we used scanning and transmission electron microscopy to observe the phagocytosis of bacteria by the protozoan, confirming the strong association between both cells and the growth dependence of T. didelphidis upon E. coli. After adherence to the protozoan surface, the bacteria induced the appearance of crater-like depressions and the ingested bacteria were intracellularly degraded.

Growth kinetic study of Tetratrichomonas didelphidis isolated from opossum Lutreolina crassicaudata and interaction with a prokaryotic cell.

Tetratrichomonas didelphidis is a flagellate protozoan found in the intestine, cecum and colon of opossums, Didelphis marsupialis. This work reports the occurrence of T. didelphidis in another opossum species, Lutreolina crassicaudata. The strain was cultivated in monoxenic culture with Escherichia coli in Diamond (TYM) medium without maltose and with starch solution (trypticase-yeast extract-starch), pH 7.5 at 28 degrees C. The growth kinetic study of T. didelphidis showed a longer time of growth and a higher number of trophozoites when inoculated with E. coli than in axenic cultures, in aerobiosis as well as under anaerobic conditions. Scanning electron microscopy showed that the bacteria adhered throughout the protozoan body and probably evoked endocytic channels, strongly suggesting the existence of endocytosis of rods by T. didelphidis. Our preliminary results suggest that the in vitro culture of T. didelphidis depends on E. coli as a growth-promoting partner, and requires monoxenic cultivation.

DNA fluorescent stain accumulates in the Golgi but not in the kinetosomes of amitochondriate protists.

Hindgut symbiotic trichomonads (uninucleate Caduceia versatilis, and multinucleate Stephanonympha sp. and Snyderella tabogae) from the dry-wood-eating termite Cryptotermes cavifrons (Kalotermitidae) accumulate DAPI (4,6diamidino-2-phenylindole) in the membranous sacs of the Golgi complex. This form of Golgi complex, typical of protists in the class Parabasalia, is called a parabasal body. Trichomonads contain organellar systems, mastigonts, that consist of four undulipodia (e.g. eukaryotic flagella and cilia), axostylar microtubules, a parabasal body and other structures. These cells bear from one (in the case of Caduceia) to hundreds (in the case of Snyderella) of mastigonts. These features are characteristic of their protist class (Parabasalia). The nuclei of all three species stained with DNA-specific stains: DAPI, SYTOX, acridine orange, propidium iodide, ethidium bromide and Feulgen, at optimal concentrations, but kinetosomes failed to stain at all. The nuclei, parabasal bodies and symbiotic bacteria (but no microtubular structures) fluoresced in glutaraldehyde-fixed cells stained with 1.45 microM DAPI. Parabasal bodies of Snyderella and Caduceia treated to remove lipids with Triton X-100, or treated with 5% trichloroacetic acid, lacked DAPI-fluorescence. I conclude that DNA, present as expected in nuclei and bacterial symbionts, is absent from and not associated with calonymphid kinetosomes. The reason for DNA-RNA stain accumulation in the Golgi cistemae is not clear.

Ethidium bromide: a fast fluorescent staining procedure for the detection of symbiotic partnership of flagellates and prokaryotes.

The hindgut of 'lower' termites harbors a dense population of flagellates and bacteria. The flagellates possess ecto- and endosymbiotic prokaryotes. Most of them are hardly visible in the phase contrast microscope. Staining with the DNA-intercalating agent ethidium bromide visualizes the nuclei of the flagellates as well as the ecto- and endosymbiotic bacteria as red objects. Furthermore, it is possible to distinguish between endosymbiotic methanogens and other bacteria. Following UV excitation, the blue-green autofluorescence of the methanogenic bacteria eclipses the red fluorescence light of the intercalated ethidium bromide. The dye facilitates the observation of symbiotic bacteria and helps identify the number, shape, localization, and dividing status of the nuclei. Thus, it is a powerful tool for the examination of microorganisms in complex habitats, which are rich in strongly autofluorescent material, like wood.