Inhibition of the Rumen Ciliate Entodinium caudatum by Antibiotics

Abstract

Axenic cultures of free-living aerobic ciliates, such as Tetrahymena thermophila and Paramecium aurelia, have been established and routinely used in laboratory research, greatly facilitating, or enabling characterization of their metabolism, physiology, and ecology. Ruminal protozoa are anaerobic ciliates, and they play important roles in feed digestion and fermentation. Although, repeatedly attempted, no laboratory-maintainable axenic culture of ruminal ciliates has been established. When axenic ciliate cultures are developed, antibiotics are required to eliminate the accompanying bacteria. Ruminal ciliates gradually lose viability upon antibiotic treatments, and the resultant axenic cultures can only last for short periods of time. The objective of this study was to evaluate eight antibiotics that have been evaluated in developing axenic cultures of ruminal ciliates, for their toxicity to Entodinium caudatum, which is the most predominant ruminal ciliate species. Scanning and transmission electron microscopy (TEM) showed that the antibiotics damaged both the cell surface and nuclei of E. caudatum and increased accumulation of intracellular glycogen. Combinations of the three least toxic antibiotics failed to eliminate the bacteria that are present in the E. caudatum culture. The combination of ampicillin, carbenicillin, streptomycin, and oxytetracycline was able to eliminate all the bacteria, but the resultant axenic E. caudatum culture gradually lost viability. Adding the bacterial fraction (live) separated from an untreated E. caudatum culture reversed the viability decline and recovered the growth of the treated E. caudatum culture, whereas feeding nine strains of live bacteria isolated from E. caudatum cells, either individually or in combination, could not. Nutritional and metabolic dependence on its associated bacteria, accompanied with direct and indirect inhibition by antibiotics, makes it difficult to establish an axenic culture of E. caudatum. Monoxenic or polyxenic cultures of E. caudatum could be developed if the essential symbiotic partner(s) can be identified.

Extending Burk Dehority’s Perspectives on the Role of Ciliate Protozoa in the Rumen

“…Glycogen accumulation in En. caudatum was associated with inhibition by antibiotics (Park et al, 2017). Glycogenesis was probably an indicator, not a cause.…”

“…For example, En. caudatum lacks a hydrogenosome (Park et al, 2017) and does not consistently shift fermentation toward butyrate as shown with mixing monofaunated cultures (Zeitz et al, 2013). Defaunation in vivo is typically associated with a decrease in molar ratio of butyrate (Newbold et al, 2015).…”

“…Although many researchers have attempted to develop protozoal axenic cultures (having no influence by prokaryotes, which contrasts with protozoal monocultures), no laboratory maintainable axenic cultures have been established (Park et al, 2017). The lack of an axenic culture of any ruminal ciliate species has hindered understanding of their metabolism, physiology, and ecology; thus, their actual roles in the rumen could only be inferred from indirect evidence (Coleman, 1962;Hino and Kametaka, 1977;Onodera and Henderson, 1980;Fondevila and Dehority, 2001b).…”

“…Live bacteria are required as sources of essential nutrients that cannot be produced or obtained from other sources (Fondevila and Dehority, 2001b;Park et al, 2017). In addition to endosymbionts, live bacteria (and possibly archaea) might be sources of prey or symbionts for recurrent recruitment.…”

“…We questioned if protozoal autolysis in the rumen is less than that projected from cultures (Diaz et al, 2014). We also questioned if ruminal ciliates can be cultured without prokaryotes (Park et al, 2017;Park and Yu, 2018a). Of course, protozoal cultures have led to critical advances in our understanding of the growth rate and function of various species, but Dr. Dehority always conditioned observations made in vitro.…”