Optimizing in vitro cultivation of Haemonchus contortus larvae: Comparative assessment of growth conditions

Haemonchus contortus, a highly pathogenic nematode of small ruminants, causes substantial economic losses worldwide. The transition from the infective third-stage larva (L₃) to the metabolically active fourth-stage larva (L₄) is critical for parasite survival, as L₄ larvae initiate blood-feeding within the host. This study aimed to optimize in vitro cultivation conditions for L₄ larvae and evaluate their metabolic activity, providing a basis for future research on parasite biology and anthelmintic development. We systematically tested six culture media (BME, DMEM, IMDM, RPMI 1640, M199, LB broth; LB without FBS) supplemented with antibiotics including gentamicin (significantly improved L₄ development: 85 % vs 70 % without, p < 0.05) to determine optimal L₃-to-L₄ conditions. Larval survival was assessed by motility under inverted microscopy; L₄ development by exsheathment, body thickening (600-800 μm), and genital primordium visibility. To assess metabolic status, we probed proteolytic activity, which represents a key function of blood-feeding larvae. A fluorogenic peptide substrate was employed to detect significant cysteine protease activity, serving as a marker of a competent digestive system. These findings establish a refined methodology for in vitro cultivation of L₄ larvae and highlight their suitability for functional studies and drug screening. By optimizing culture conditions and demonstrating active proteolysis, this study provides a valuable platform for investigating stage-specific parasite biology and identifying novel therapeutic targets.