Metabolic Profile of Histomonas meleagridis in Dwyer's Media with and Without Rice Starch

Abstract

Background and objectives: Histomonas meleagridis, the causative agent of histomonosis (i.e., blackhead disease), threatens the poultry industry with serious economic losses due to its high mortality and morbidity in turkey and chicken flocks. In vitro studies are complicated by the inability to culture the parasite axenically. Histomonas meleagridis has been propagated in Dwyer's media, which contains a starch source and serum, for over 50 years. The presence of insoluble starch component in Dwyer's media represents an obstacle for the commercialization of such media, and the role of starch in media is poorly understood.

Methods: To investigate the intracellular metabolomic differences in H. meleagridis and undefined bacteria grown in Dwyer's media with rice starch (SD) and without rice starch (NR), we conducted a global metabolomics analysis using ultra-high-performance liquid chromatography-high-resolution mass spectrometry.

Results: SD significantly supported the growth of H. meleagridis compared to NR. There was no significant difference in bacterial growth between SD and NR media at various timepoints. From the intracellular metabolic analysis of samples collected from the SD and NR media, a total of 170 known metabolites were identified. H. meleagridis appears to be the major contributor to global metabolic differences.

Conclusions: We found that riboflavin had the highest variable importance in the projection score, and metabolites involved in riboflavin biosynthesis significantly contributed to the differences between SD and NR in the media immediately after the inoculation of H. meleagridis and undefined bacteria, warranting further investigations into the role of riboflavin biosynthesis in H. meleagridis growth.

Keywords: Histomonas meleagridis; mass spectrometry; metabolomics; protozoa propagation; riboflavin.

Figure 1

Growth curve of Histomonas meleagridis grown in Dwyer’s media with (SDM) and without (NR) rice starch. The mean of the log values is represented on the vertical axis and the hours post inoculation (HPI) on the horizontal axis.

Figure 2

Growth curve of undefined bacteria in Histomonas meleagridis cultures using Dwyer’s media with (SDM) and without (NR) rice starch. The mean of the log values is represented on the vertical axis and the hours post inoculation (HPI) on the horizontal axis. “CFU” stands for colony-forming unit.

Figure 3

The volcano plots show that the intracellular metabolites are statistically and significantly different between Histomonas meleagridis and undefined bacteria in Dwyer’s media with (SD) and without (NR) rice starch. Fold change equals log₂ (average relative abundance for NR/average relative abundance for SD). Red indicates that the metabolite has higher relative abundance in NR treatment, while blue indicates the metabolite has lower abundance in NR treatment. The metabolites were collected immediately at timepoint t0—representing ~0.25 h post infection—after the inoculation of H. meleagridis.

Figure 4

Partial least squares discriminant analysis (PLS-DA) of metabolites in Dwyer’s media with (SD) and without (NR) rice inoculated with Histomonas meleagridis and undefined bacterial population at blank and 0 HPI (t0). Ellipse represents 95% confidence interval.

Figure 5

Variable importance in projection (VIP) scores for the top 15 metabolites contributing the most to the differences in the metabolic profile between Dwyer’s media with and without rice inoculated with Histomonas meleagridis and undefined bacteria in the media blank and 0 HPI. Metabolites with a VIP score over 1 drive the separation in the PLS-DA plot. Riboflavin has the highest VIP score in all 5 components (5.3907-1.1438).

Figure 6

The pathway analysis reveals that riboflavin metabolism and pathways generating metabolic precursors of riboflavin are significantly altered based on media composition. All metabolites with variable importance in projection (VIP) scores >1 were used to conduct a pathway analysis to identify changes in metabolic pathways altered in NR media compared to SD media. Each circle represents a pathway, and the colors indicate the significance (y-axis), while the size depicts the pathway impact (x-axis). The more intense the shade of red, the lower the p-value, and the bigger the circle, the higher the impact of the pathway. Only pathways with p < 0.05 are labeled.

Figure 7

Riboflavin and metabolic precursors from interconnected metabolic pathways are altered by the omission of rice starch. G6P: glucose-6-phosphate, TCA: tricarboxylic citric acid, sedoheptulose 1,7P: sedoheptulose 1,7 phosphate, Ru5P: Ribulose 5-Phosphate, Ribo-5P: Ribose 5-phosphate, AMP: adenosine monophosphate, GTP: guanine triphosphate, FMN: flavin mononucleotide, FAD: flavin adenine dinucleotide. The metabolites in italics have increased in levels in the NR media compared to the SD media.