Efficacy of flukicides on Fasciola gigantica, a food-borne zoonotic helminth affecting livestock in Bangladesh

Abstract

Fasciola gigantica, the causative agent of tropical fasciolosis, is a food-borne zoonotic trematode that affects around 80% livestock of Bangladesh. Triclabendazole (TCBZ), nitroxynil (NTON) and oxyclozanide (OCZN) are frequently used against fascioliasis; however, the current status of potency of these flukicides was unknown. In this study, in vitro efficacy of TCBZ, NTON and OCZN at various concentrations on F. gigantica has been evaluated by relative motility (RM), morphological distortions of apical cone through an inverted microscope, architectural and ultra-structural changes through histopathological and scanning electron microscopy (SEM). It is observed that TCBZ, NTON and OCZN at higher concentrations significantly (P < 0.05) reduced RM of the flukes compared to untreated control. NTON at 150 μg mL⁻¹ was the most potent to reduce the motility within 4 h whereas TCBZ and OCZN were much delayed. Histopathological changes showed swollen, extensive cracking, numerous vacuoles and splitting of the tegument surrounding the spines; spine dislodged from its socket in treated flukes compared to untreated worms. Histopathological changes were more conspicuous at higher doses of TCBZ, NTON and OCZN. SEM has shown the disruption of the apical cone, apart from swelling of the tegument on the ventral surface corrugation and disruption of the ventral apical cone. All these changes indicate that NTON is the most potent in killing flukes in vitro among the tested flukicides and suggest the presence of TCBZ-resistant fluke populations in Bangladesh. It is imperative to explore the in vivo effects of these flukicides and subsequently their molecular mechanisms.

Keywords: Bangladesh; Fasciola gigantica; flukicides; food-borne trematode; in vitro; scanning electron microscopy.

Graphical abstract

Fig. 1.

RM rate of Fasciola gigantica treated with various concentrations of TCBZ (A), NTON (B) and OCZN (C) in vitro method.

Fig. 2.

Stereomicroscopic figures showing the histopathology of the tegument of F. gigantica. (a) Control F. gigantica incubated in RPMI-1640 medium for 8 h, showing the tegument with normal appearances of parenchyma (P), spines (S) embedded in the intact tegument (T) and muscle layers (M) lying underneath the basement membrane (Ba). Flukes are treated with TCBZ at 10 μg mL⁻¹; NTON at 50 μg mL⁻¹ and OCZN at 0.02 μg mL⁻¹ for 8 h of incubation, showing the formation of small blebs (BL) in the surface of tegument (T), mild separation of tegument between the spines (S) and underlying tissue (T) and dislodged spines (DS) in the micrograph of (b), (e) and (h). Flukes are treated with TCBZ at 20 μg mL⁻¹; NTON at 100 μg mL⁻¹ and OCZN at 0.2 μg mL⁻¹ for 8 h of incubation, showing the small blebs (BL), disruption of blebs (DL) on the tegument and formation of small vacuoles (SS) in the cytoplasm, while spine (S), muscle (M) lying the underneath the basement membrane (Ba) in the micrographs of (c), (f) and (i). At high concentration of TCBZ at 40 μg mL⁻¹, NTON at 150 μg mL⁻¹ and OCZN at 2 μg mL⁻¹, showing the formation of more outward small vacuoles (SS), small blebs (BL), degeneration and sloughing of tegument and disruption of blebs (DL) in the micrograph of (d), (g) and (j), respectively.

Fig. 3.

SEM images of the control and treated flukes. (A) SEM of apical cone surface of control flukes showing smooth ventral sucker (VS) with thick rims (R) covered with transverse folds (T) and appear spineless. SEM of the ventral sucker (B), (E) and (H) showing tegumental blebbing (TB), little disruption (L) apart from the swelling of tegument (T) on the ventral surface at a concentration of TCBZ at 10 μg mL⁻¹; NTON at 50 μg mL⁻¹ and OCZN at 0.02 μg mL⁻¹ for 8 h of incubation. Pronounced tegumental swelling (TS) and disruption (L) and distortion of the ventral suckers (VS) showed in the SEMs (C), (F) and (I) during flukes were treated with CBZ at 20 μg mL⁻¹; NTON at 100 μg mL⁻¹ and OCZN at 0.2 μg mL⁻¹ concentrations. SEM of flukes treated with TCBZ at 40 μg mL⁻¹, NTON at 150 μg mL⁻¹ and OCZN at 2 μg mL⁻¹ concentrations showing relatively little disruption (L) on the ventral surface of flukes, apart from the swelling of the tegument (T) and swollen rim (R) of ventral sucker.

Fig. 4.

(A) SEM showing smooth and flourish wall of the ventral sucker (VS) of control fluke at higher magnification (500×). (B), (C), (E), (F), (H) and (I) SEM of the ventral sucker of flukes treated with TCBZ at 10 μg mL⁻¹, TCBZ at 20 μg mL⁻¹, NTON at 50 μg mL⁻¹, NTON at 100 μg mL⁻¹, OCZN at 0.2 μg mL⁻¹ and OCZN at 0.2 μg mL⁻¹ showing severe tegumental distortion (arrow marks) and sloughing (L) but all these lesions were more pronounced at the concentrations of TCBZ at 40 μg mL⁻¹, NTON at 150 μg mL⁻¹ and OCZN at 2 μg mL⁻¹ in the SEM (D), (G) and (J).

Fig. 5.

(A) SEM micrograph of tegument (T) surface in control flukes shows normal appearance. (B–D) SEM of tegument of treated flukes with various concentrations of TCBZ showing gradual distortion of spines. (E–G) SEM of treated flukes with various concentrations of NTON showing gradual distortion of spines and blebbing of tegument. (H–J) SEM of treated flukes with various concentrations of OCZN showing severe damaged (arrow marks) of tegument and gradual distortion of spines (S).

Fig. 6.

(A) SEM image of spines (S) of control fluke shows finger-like protrusion at their tips. (B–J) SEM micrographs of treated flukes with various concentrations of TCBZ, NTON and OCZN showing extensive distortion (arrow marks) of the tegument (T) with losses of numerous spines (S). Severe tegumental distortion was found in flukes treated with higher concentration (150 μg mL⁻¹) of NTON (G).