Fig 1.
The oocyst excretion pattern, intensity of diarrhea and body weight gains of the GB piglets orally challenged with TU502 oocysts.
(A) Daily oocyst shedding (means ± SEM) in feces. The rectal swabs were processed and counted in 30 microscopic fields under 1,000x magnificent. (B) Daily diarrhea scores (means ± SEM) observed in piglets. Symptoms were monitored and scored daily by 2 individuals; diarrhea was scored from 0 to 4 (0 –no diarrhea; 1 –brown, grey, soft stool, mild diarrhea; 2 –brown yellow, mucoid, mild to moderate diarrhea; 3 –yellow, mucoid to watery, moderate diarrhea; 4 –white to yellow, watery, severe diarrhea). Two-way ANOVA with Sidak's multiple comparisons test was performed using GraphPad Prism 7.01. *p<0.05, **p<0.01, ****p<0.0001. TU502-infected group (n = 10, closed circle ●); Uninfected control (n = 4, open circle ○). Loose feces in uninfected animals, attributed to the milk diet, are often observed.
Fig 2.
Accumulated counts of oocyst excretion in feces of TU502-challenged piglets treated with either, AZR, NTZ or AZR+NTZ, as compared with an untreated infected group.
There was a notable contrasting in the level of oocyst excretion between days 1–5 (A) and day 6–10 (B) of treatment, as shown with box-and-whiskers plot (min to max). Mann-Whitney test was conducted using GraphPad Prism 7.01. *p<0.05, **p<0.01.
Fig 3.
Daily diarrhea scores observed in piglets treated with either AZR (A), NTZ (B) or AZR+NTZ (C), compared with uninfected AZR+NTZ-treated and untreated groups (D). Two-way ANOVA with Tukey's multiple comparisons test was conducted using GraphPad Prism 7.01. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. TU502-infected group (n = 10, closed circle ●) was compared with AZR-treated group (n = 5, closed squire ■) in A; with NTZ-treated group (n = 5, closed triangle ▲) in B; and with AZR+NTZ-treated group (n = 7, closed rhombus ◆) in C; Uninfected group treated with AZR+NTZ (n = 5, open rhombus ◇) and untreated control group (n = 4, open circle ○) are presented in D.
Fig 4.
Gaining of body weight after drug treatment for C. hominis-infected piglets.
Gnotobiotic piglets were inoculated orally with C. hominis oocysts two days after birth and treated with AZR, NTZ, or combined AZR and NTZ three days post challenge. Body weight was measured daily. Two-way ANOVA with Tukey’s multiple comparisons test was conducted using Graphpad Prism 7.01. * p<0.05. C. hominis TU502-infected group (n = 7, closed circle ●); TU502-infected, AZR-treated group (n = 3, closed squire ■); TU502-infected, NTZ-treated group (n = 3, closed triangle ▲). The partial measurement from a piglet, which was dead on day 8 post NTZ treatment were included; TU502-infected, AZR+NTZ-treated group (n = 7, closed rhombus ◆); Uninfected, AZR+NTZ treated group (n = 3, open rhombus ◇); and untreated control group (n = 2, open circle ○). The C. hominis TU502-infected group and untreated control group already shown in Fig 1 were added for comparison. The measurement of body weight from experiment I, II, and III was excluded due to the inaccuracy of scale.
Fig 5.
Microscopic lesions of C. hominis infection.
Photomicrographs are from ileum and large intestine, stained with hematoxylin and eosin, magnified 400 times normal. Normal small and large intestine are shown in A and B, respectively. Small and large intestine from C. hominis are shown in C and D, respectively with moderate epithelial dysplasia, moderate loss of Goblet cells, moderate lymphocytic inflammation, and C. hominis organisms (black arrows). Small and large intestine from C. hominis treated with ARZ+NTZ are shown in E and F, respectively with mild epithelial dysplasia, mild loss of Goblet cells, and C. hominis organisms (black arrows). The overall pathology score is shown in G, with component scores for dysplasia/inflammation and Goblet cells shown in H and I, analyzed by the Mann-Whitney t-test with p<0.05. Dashed lines indicate the average score for uninfected piglets.
Table 1.
Concentration of AZR and NTZ, plus metabolites TIZ and TIZ-glucuronide in sera and rectal swabs.
Table 2.
Serum biochemical data and hematology of gnotobiotic piglets used in this study.