Optimizing Balantioides coli in vitro maintenance: effects of temperature and lipid supplementation in modified Pavlova medium

Balantioides coli is a ciliated protozoan whose main hosts include pigs, non-human primates, and humans. Balantidiasis is a zoonotic disease capable of causing clinical dysentery in both animals and humans. Laboratory diagnosis is traditionally based on microscopic parasitological techniques, and only a few studies have explored in vitro culture for parasite isolation and maintenance, mainly due to its labor-intensive routine. This study aimed to evaluate the maintenance and growth performance of B. coli in modified Pavlova medium supplemented with different lipid sources under varying temperatures and incubation times. Between February and April 2025, a B. coli strain previously isolated from pig feces and maintained in the laboratory was analyzed. A standard inoculum of 500 trophozoites was transferred into culture tubes containing Pavlova medium prepared under four conditions: without serum (P0), supplemented with coconut water (PC), fetal bovine serum (PB), or equine serum (PE). Cultures were incubated at five temperatures (32, 34, 36, 38, and 40 °C) and assessed at four time points (24, 48, 72, and 96 h). Growth was quantified as trophozoites per mL. The highest B. coli counts occurred in media containing fetal bovine (PB) and equine (PE) serum, particularly in PE at 40 °C for 48 h. Coconut-water medium (PC) showed only a mild increase at 36–38 °C after 72–96 h, whereas the control (P0) supported limited growth. Significant differences (p < 0.05) were observed among media types, incubation periods, and temperatures, except between PB and PE at 32–36 °C. Modified Pavlova medium supplemented with fetal bovine or equine serum, particularly under incubation at 38–40 °C, provided the most favorable conditions for B. coli growth and maintenance. This study provides, for the first time, a systematic evaluation of lipid supplementation and incubation conditions, establishing standardized parameters for the routine laboratory cultivation of this zoonotic ciliate.