Establishment of a stable monoculture system for <i>Entodinium furca monolobum</i> and isolation of <i>Escherichia</i> spp. as growth-promoting bacteria.

Abstract

<h4>Introduction</h4><i>Entodinium furca monolobum</i> is a common rumen ciliate protozoan, while it remains poorly characterized due to the lack of a stable monoculture system.<h4>Methods</h4>In the present study, we established a stable <i>in vitro</i> monoculture of <i>Ent. furca monolobum</i> using a modified SP medium with inoculum derived from Holstein cows through filtration-based enrichment and serial dilution. We isolated bacteria from the monoculture supernatant and ciliate-associated fractions, and evaluated candidate isolates in co-culture assays. We also profiled bacterial communities by amplicon sequencing.<h4>Results</h4>A stable <i>in vitro</i> monoculture of <i>Ent. furca monolobum</i> was established. Eight bacterial species were isolated from the monoculture supernatant and four from the ciliate-associated community. <i>Escherichia coli and Escherichia fergusonii</i> were consistently detected in both fractions and significantly promoted ciliate proliferation, with <i>E. coli</i> showing a stronger effect (<i>p</i> < 0.05). Community profiling showed distinct bacterial structures between monoculture supernatant and protozoa-free rumen fluid, and Gammaproteobacteria dominated in <i>E. coli</i>-supplemented cultures.<h4>Discussion</h4>This work not only overcomes the long-standing challenge of maintaining <i>Ent. furca monolobum</i> <i>in vitro</i> culture, but also provides a methodological framework for developing defined monoxenic systems. The stable monoculture system and identification of growth-promoting <i>Escherichia spp.</i> provide a methodological basis for mechanistic studies of protozoan-bacterial interactions in the rumen.