Fig 1.
Experimental design of the studied groups.
Fig 2.
SEM micrographs of the prepared NPs showing regular, smooth and spherical shape.
(A) CS/Alg NPs (X 35,000). (B) Spiramycin loaded CS/Alg NPs (X 55,000). (C) Propolis loaded CS/Alg NPs (X 55,000). (D) Spiramycin/propolis loaded CS/Alg NPs (X 55,000).
Fig 3.
TEM micrographs of the prepared NPs revealing increase in particle size upon drug loading (X 55,000).
(A) CS/Alg NPs. (B) Spiramycin loaded CS/Alg NPs. (C) Propolis loaded CS/Alg NPs. (D) Spiramycin/propolis loaded CS/Alg NPs.
Table 1.
Zeta-potential (ΞΆ) and entrapment efficiency (EE) of the prepared nano formulae.
Fig 4.
Kaplan-Meier overall survival curve for all subgroups.
Table 2.
Comparison between all subgroups regarding mice mean survival time and mortality rate on the eighth day post-infection.
Fig 5.
Comparison between the different studied subgroups according to the mean parasite count in liver, spleen and brain.
Fig 6.
Giemsa-stained impression smears of liver, spleen and brain revealing T. gondii tachyzoites.
(A) Toxoplasma tachyzoites in infected untreated control liver (Giemsa stain X 1000). (B) Toxoplasma tachyzoites in infected treated liver (Giemsa stain X 1000). (C) Toxoplasma tachyzoites in infected untreated control spleen (Giemsa stain X 1000). (D) Toxoplasma tachyzoites in infected treated spleen (Giemsa stain X 1000). (E) Toxoplasma tachyzoites in infected untreated control brain (Giemsa stain X 1000). (F) Toxoplasma tachyzoites in infected treated brain (Giemsa stain X 1000).
Table 3.
Parasite count and percent reduction in liver, spleen and brain among the different studied subgroups.
Fig 7.
SEM of Toxoplasma tachyzoites.
(A) Tachyzoite from subgroup I b (infected untreated control), revealing crescent shape with apparent conoid (X 10,000). (B) Tachyzoite from subgroup II a, showing evident loss of its smooth surface with erosion and protrusion of the surface. (X 10,000). (C) Tachyzoite from subgroup II b, showing shrunken organism losing its crescent shape, smooth surface, conoid and tapered ends with profound furrows and ridges (X 10,000). (D) Tachyzoite from subgroup II c, showing a shrunken distorted parasite losing the crescent shape, tapered ends and conoid with noticeable surface erosion and ulceration (X 10,000). (E) Tachyzoites from subgroup II d, showing loss in the crescent shape, smooth surface, conoid and tapered ends with multiple blebs on the parasite surface (X 10,000). (F) Tachyzoites from subgroup II e, showing a rupturing parasite losing its smooth surface (X 10,000). (G) and (H) Tachyzoites from subgroup II f, showing disfigured rupturing organism losing its conoid with disorganised lacerated surface (X 10,000).
Fig 8.
H&E-stained sections of hepatic tissues in different studied groups.
Infected untreated group: (A) Severe capsular oedema and dense inflammatory exudate is seen on liver capsule (c) and diffuse severe portal inflammation (P). (B) Portal tract (p) showing severe mononuclear inflammation with interface hepatitis (black arrow) and marked vascular congestion. (C) Large mononuclear lobular inflammation focus showing necrotic hepatocytes and numerous parasites (yellow arrow). Spiramycin treated group: (D) Moderate capsular oedema with mild inflammatory exudate is seen on liver capsule (c) and moderate inflammation is seen in portal tracts (P). (E) Portal tract (p) showing moderate mononuclear inflammation with interface hepatitis (black arrow) and moderate vascular congestion. (F) Mononuclear lobular inflammation showing necrotic hepatocytes. Propolis treated group: (G) Moderate capsular oedema with mild inflammatory exudate is seen on liver capsule (c) and moderate/severe inflammation is seen in portal tracts (P). (H) portal tract (p) showing moderate mononuclear inflammation with interface hepatitis (black arrow) and marked vascular congestion. (I) Large mononuclear lobular inflammation focus showing necrotic hepatocytes. CS/Alg NPs treated group: (J) Moderate capsular oedema with mild inflammatory exudate is seen on liver capsule (c). Moderate/severe inflammation is seen in portal tracts (P). (K) Portal tract (p) showing moderate mononuclear inflammation with interface hepatitis (black arrow) and marked vascular congestion. (L) Large mononuclear lobular inflammation focus showing necrotic hepatocytes. Spiramycin CS/Alg NPs treated group: (M) Mild capsular oedema with no exudate on liver capsule (c). Moderate inflammation is seen in portal tracts (P). (N) Portal tract (p) showing moderate mononuclear inflammation with focal interface hepatitis (black arrow) and moderate vascular congestion. (O) Small mononuclear lobular inflammation focus showing few necrotic hepatocytes. Propolis CS/Alg NPs treated group: (P) Mild capsular oedema with no exudate on liver capsule (c). Minimal inflammation is seen in portal tracts (P). (Q) Portal tract (p) showing mild mononuclear inflammation with focal interface hepatitis (black arrow) and moderate vascular congestion. (R) Small mononuclear lobular inflammation focus showing few necrotic hepatocytes. Spiramycin/Propolis loaded CS/Alg NPs treated group: (S) Normal hepatic capsule with no oedema (c). Minimal inflammation is seen in rare portal tracts (P). (T) Portal tract (p) showing minimal mononuclear inflammation with no interface hepatitis. No vascular congestion is seen. (U) Small mononuclear lobular inflammation focus is occasionally seen.
Fig 9.
Sections of splenic tissues in infected untreated group (A-B) and treated groups with different regimens (C-N).
(A) Infected untreated group showing perisplenitis (c) and severe congestion of red pulp (R) which occupied most of splenic architecture in expanse of white pulp (W). (B) High power view showing increased number of histiocytes and megakaryocytes (yellow arrows) with numerous parasites (red arrow) in infected untreated group. (C-N) Perisplenitis (C), red pulp congestion (R) and the count of megakaryocytes/histiocytes (yellow arrows) improved in different degrees in the treated groups.
Fig 10.
H&E-stained brain sections of different studied groups.
(A) Infected untreated group showing mononuclear parenchymal infiltrate (black arrow) with perivascular oedema (v) (X100). (B) Mild improvement is seen in spiramycin treated group showing residual meningitis (asterisk) and perivascular oedema. (X100). (C-F) Mononuclear parenchymal infiltrate (black arrow) and perivascular oedema (v) are still noted in propolis, CS/Alg NPs, spiramycin CS/Alg NPs and propolis CS/Alg NPs treated groups respectively (X100). (G) Best results are seen in spiramycin/ propolis loaded CS/Alg NPs treated group with normal neurons, no meningitis or mononuclear infiltrate (X100).