Toxicity of antibiotics on rumen protozoan Entodinium caudatum and its associated microbes

Abstract Text: Rumen ciliate protozoa play important roles in rumen function. However, knowledge on the metabolism and physiology of rumen protozoa is limited, mostly because of lack of axenic cultures of rumen ciliate protozoa needed to generate direct evidence. Antibiotics alone and in combination with physical separation have been successfully used in generating axenic cultures of free-living aerobic ciliate protozoa, such as species of Tetrahymena and Paramecium. However, no effort has been successful in developing axenic cultures of rumen protozoa. Rumen protozoa have lived together with a high density of rumen bacteria and other groups of microbes for many millions of years, and killing of the associated bacteria and/or archaea resulted in loss of viability of rumen protozoa. The objective of this experiment was to evaluate the toxicity of different antibiotics to Entodinium caudatum, a major species of Entodinium, which as a genus accounts for more than 90% of the total rumen protozoa in cattle and sheep fed high-concentrate diets. Based on the mode of action, eight antibiotics were selected that each has a broad spectrum to kill or to inhibit bacteria. En. caudatum cells grown in a culture were filtrated and washed to remove most of the free-living bacteria and methanogens. The washed En. caudatum cells were then fed a protozoal feed containing ground wheat, alfalfa and grass and incubated for 72 hours with 4 different concentrations and three replicates per antibiotic treatment. After incubation, En. caudatum cell counts, optical density, and electron microscopy (both scanning and transmission) were used to determine En. caudatum viability, growth of contaminating bacteria, and changes of intracellular structure of En. caudatum cells. Except ampicillin, all the tested antibiotics decreased En. caudatum growth in a concentration dependent manner. Chloramphenicol appeared to be the most toxic to the viability of En. caudatum. Scanning electron microscopy and transmission electron microscopy revealed few ecto- or endo-symbiotic microbes of the En. caudatum cells irrespective of the antibiotic treatments. Damages to the intracellular structures were detected by scanning electron microscopy, especially among the En. caudatum cells treated with chloramphenicol. Transmission electron microscopy is underway to further examine cellular damages, and several antibiotics cocktails are being evaluated for their usefulness to kill the associated microbes and generate an axenic En. caudatum culture.

Keywords: Entodinium caudatum, axenic culture, electron microscopy