Rice starch

The virulent monoxenic clonal culture H. meleagridis/turkey/Austria/2922-C6/04 (Hess et al., 2006 (link)) co-cultivated with the laboratory strain E. coli DH5α (Invitrogen, Vienna, Austria) (Ganas et al., 2012 (link)) was in vitro passaged 24 times prior to infection. The inoculum consisted of H. meleagridis cells in Medium 199 with Earle’s salts, L-glutamine, 25 mM HEPES and L-amino acids (Gibco^TM Invitrogen, Austria), 15% fetal calf serum (Gibco^TM Invitrogen) and 0.25% (w/v) rice starch (Sigma-Aldrich, Vienna, Austria).

Rice starch (Sigma-Aldrich) as a biopolymer and lithium iodide salt (Aldrich, crystalline powder, 99.9% trace metal basis) were used in this work. Each sample was prepared using a solution cast technique. Rice starch (1 g) was dissolved in 25 ml of DI-water and stirred and heated to 80°C until gelatinization. After gelatinization, the solution was cooled to room temperature with continued stirring and different amounts of lithium salt (LiI) were added (5, 10, 15, 20, 25, 30 and 35 wt. %). The solution was stirred to obtain a homogenous mixture. Solutions were then cast onto Teflon petri dishes and oven dried at 60°C for 24 h. After drying, solid films were cast.
Conductivity and frequency-dependent studies were carried out using an electrochemical impedance spectroscopy (EIS) analyzer (Hioki, 3532-50 LCR HiTESTER) with a frequency range of 50 Hz to 5 MHz. The samples were compressed between two stainless steel blocking electrodes with areas of 2.98 cm². The imaginary parts of impedance were automatically computed with a LCR HiTESTER. The ionic parameter of ac-conductivity was calculated using following equation. where is the imaginary part of impedance known as dielectric loss, ω is angular frequency and is the permittivity of the free space [27] .

To study the interactions between R. bili IPLA60002 and R. gauvreauii IPLA60001 we carried out coculture combinations in M2 medium with 0.2% rice starch (Sigma-Aldrich) as sole carbon source. The coculture conditions were as follows: 10 mL of initial exponential phase cocultures of IPLA60002 and IPLA60001 (6 h, OD₆₅₀ 0.4–0.5), and 10 mL cocultures in middle exponential phase (8 h, OD₆₅₀ 0.6–0.7). As controls, the strains were grown concurrently in monoculture. Three technical replicates were performed for each condition, and cultures were inoculated from stock liquid cultures at 1% of total liquid volume. The tubes were centrifuged for 10 min at 10,000 rpm and the pellet was immediately resuspended in 1 mL of RNAlater (Qiagen) and frozen at −80°C.

Corn starch and rice starch used for the AF and ST production studies were purchased from Sigma-Aldrich (Merck Life Science Kft., Budapest, Hungary). The ratio of amylose to amylopectin in Corn starch is 27:73 w/w% (Sigma), while rice contains slightly less, 23% amylose (https://www.sigmaaldrich.com/HU/en/product/sial/s4126). All chemicals used in the study were of analytical grade.

Mice were anesthetized and submitted to intratracheal (i.t.) P. brasiliensis infection as previously described. Briefly, after intraperitoneal (i.p.) anesthesia the animals were infected with 1 × 10⁶ Pb18 yeast cells, contained in 50 µL of PBS, by surgical i.t. inoculation, which allowed dispensing of the fungal cells directly into the lungs. The skins of the animals were then sutured, and the mice were allowed to recover under a heat lamp. Groups of infected B10.A and A/J mice were treated with daily i.p. injections of 5 mg/mL of 1MT or 1 mg/mL of rice starch (Sigma-Aldrich) as control. Mice were sacrificed after 96 h or 2 weeks of infection.

The inocula used for vaccination or infection originated from a clonal culture of H. meleagridis/Turkey/Austria/2922-C6/04, established from cecal contents of a diseased turkey (19 (link)). The clonal culture was propagated for a short period of time (21 passages) to preserve virulence, hence used for challenge, or for 295 passages to achieve attenuation, according to previous protocols (5 (link), 20 (link)). The parasites were kept as cryopreserved cultures (stored at −150°C) were then thawing and incubated at 40°C in a culture medium consisting of Medium 199 supplemented with Earle’s salts, l-glutamine, 25 mM HEPES and l-amino acids (Gibco™, Invitrogen, Lofer, Austria), 15% of fetal calf serum (Gibco™) and 0.66 mg of rice starch (Sigma–Aldrich, Vienna, Austria). Before administration, the number of viable cells was determined using a Neubauer cell counting chamber (Reichert, Buffalo, NY, USA) after staining with 0.4% trypan blue solution and adjusted to 1 × 10⁶ histomonads per ml of medium.