الفهرس 
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Abstract
Summary
Cryptosporidiosis is a ubiquitous infectious disease, caused by the protozoan parasites Cryptosporidium parvum and Cryptosporidium hominis, causing acute persistent, and chronic diarrhea with fatal consequences in immunocompromised individuals. Due to the absence of effective drug treatments, vaccine development that prevents disease or reduces the severity and longevity of infection is important.
In the present study, the effectiveness of Nitazoxanide (NTZ) or / and, the protective role of C. parvum oocysts crude antigen (vaccine) were evaluated in C. parvum infected immunocompetent and immunosuppressed mice groups.
Our study was conducted on 160 laboratory male, Swiss albino mice CD-1 strain, aged 6-7 weeks with a weight range of 20-25 gm. and were divided to three main groups.
Control group (A) comprising 10 mice were left (unvaccinated, uninfected and untreated) and considered as negative control group. group (B) immunocompetent infected groups comprising 50 mice were orally inoculated with10000 C. parvum oocysts/mouse. group (C) immunosuppressed groups Comprising 100 mice received orally (Dexamethasone) drug in a dose of 0.25µg/g/day for 14 successive days prior to inoculation with C. parvum oocysts. The immunosuppressed mice continued to receive Dexamethasone at the same dose throughout the experiment.
group (B) was subdivided into 5 equal subgroups. Subgroup (B1) infected, un-treated while subgroup (B2) infected, seven days post infection, treated with Nitazoxanide (NTZ) for seven successive days. Subgroup (B3) vaccinated with three successive boosters, and left uninfected and untreated. Subgroup (B4) vaccinated, infected after 3days from last immunization. Subgroup (B5) vaccinated, infected, then treated with Nitazoxanide (NTZ) for seven successive days.
group (C) was subdivided into 10 equal subgroups. Subgroup (C1) immunosuppressed (uninfected – untreated). Subgroup (C2) immunosuppressed then infected- untreated mice received orally (Dexamethasone) drug in a dose of 0.25µg/g/day for 14 successive days, then orally inoculated with10000 C. parvum oocysts/mouse. Subgroup (C3) immunosuppressed, infected, then treated with Nitazoxanide (NTZ). Subgroups that received vaccine after immunosuppression; subgroup (C4) immunosuppressed then vaccinated. Subgroup (C5) immunosuppressed, vaccinated, then infected. Subgroup (C6) immunosuppressed, vaccinated, infected then treated with (NTZ). Subgroups that received vaccine before immunosuppression; subgroup (C7) vaccinated. Subgroup (C8) vaccinated then immunosuppressed group. Subgroup (C9) vaccinated, immunosuppressed, then infected. Subgroup (C10) vaccinated, immunosuppressed, infected, then treated with Nitazoxanide (NTZ).
Fecal analysis was performed and the number of C. parvum oocysts/gm of feces was counted for parasitological studies 21 days post infection. Serological studies detecting sera antibody IgG, IgM, and IgA levels were performed. histopathological examination of intestinal sections was held.
In immunocompetent infected groups, the highest percentage of reduction in oocysts shedding was found in vaccinated, infected then NTZ treated mice group (B5) and in vaccinated, then infected mice group (B4) which were very highly and highly significant (P <0.001 & 0.01) respectively. Followed by infected then NTZ treated group (B2), where the percentage of reduction was significant (P <0.05).
In immunosuppressed infected groups, it was also found that the highest percentage of reduction in oocysts shedding was found in vaccinated, immunosuppressed, infected then NTZ treated mice group (received vaccine before immunosuppression) (C10) and in immunosuppressed, vaccinated, infected then treated with NTZ group (received vaccine after immunosuppression) (C6) which were very highly significant (P <0.001). Comes next, vaccinated, immunosuppressed, then infected group (receive vaccine before immunosuppression) (C9) and immunosuppressed, vaccinated, then infected group (receive vaccine after immunosuppression) (C5), they were very highly and highly significant (P < 0.001 & 0.01) respectively. Followed by immunosuppressed, infected, then treated with NTZ mice group (C3), the percentage of reduction was significant (P <0.05).
The mean sera levels of specific anti-Cryptosporidium IgG, IgM and IgA for immunocompetent groups, 21day post infection, was increased significantly in all groups (P <0.001). The highest mean levels were detected in B1 (infected – untreated) then B4 (vaccinated, then infected) group, followed by B3 (vaccinated) group compared to uninfected - untreated control mice group (group A). Compared to infected – untreated (B1) group, the highest percentage of reduction (P <0.001) in sera mean levels of IgG, IgM and IgA antibodies was detected in group B5 that received vaccine, infection, then treated with NTZ, and in group B2 (infected then treated with NTZ). Significant reduction was found in sera IgG and IgA mean levels in group B3 (vaccinated); also, significant reduction (P <0.05) was found in sera IgG in group B4 (vaccinated then infected). Insignificant reduction (P >0.05) in mean sera IgM levels was found in B3 (vaccinated) and B4 (vaccinated then infected) groups. Insignificant reduction (P >0.05) in sera IgA mean levels was found in group B4 (vaccinated then infected). In conclusion group B5 that received vaccine, infection, then treated with NTZ showed the highest improvement of the immune status and were closer to recovery.
The mean sera levels of Specific anti-Cryptosporidium IgG, IgM and IgA in immunosuppressed groups, 21day post infection, was increased significantly (P <0.001) in all groups except group C1 (immunosuppressed). The immunocompetent groups showed better immune response than immunosuppressed groups. The highest mean antibody levels (P <0.001) were detected in group C5 (immunosuppressed, vaccinated, then infected) and group C9 (vaccinated, immunosuppressed, then infected) compared to normal control group A (uninfected - untreated). Compared with C2 group (immunosuppressed infected-untreated), a statistically significant reduction (P <0.05) in the mean IgG& IgA sera levels was observed in group C3 (immunosuppressed, infected, then treated with NTZ). Statistically insignificant reduction (P >0.05) in the mean IgM sera levels was observed in group C3 (immunosuppressed, infected, then treated with NTZ).
Regarding the mean sera IgG level; vaccination after immunosuppression caused a highly statistically significant reduction (P <0.001) in, that was observed in group C4 (immunosuppressed then vaccinated). The mean IgG sera level in group C5 (immunosuppressed, vaccinated, then infected) showed highly significant increase (P <0.01). In addition, statistically insignificant reduction (P >0.05) was observed in group C6 (immunosuppressed, vaccinated, infected, then treated with NTZ). On the other hand, there was significant reduction (P <0.05) between the mean IgG sera levels in C5 (immunosuppressed, vaccinated, then infected) & C6 groups (immunosuppressed, vaccinated, infected, then treated with NTZ) which mean that there was improvement in immune status.
Vaccination before immunosuppression caused a statistically significant reduction (P <0.05) in the mean sera IgG level in group C8 (vaccinated then immunosuppressed). A highly statistically significant increase (P <0.01) was observed in the mean IgG sera level in group C9 (vaccinated, immunosuppressed, then infected). Statistically significant reduction (P <0.05) was observed between the mean IgG sera levels in groups C10 (vaccinated, immunosuppressed, infected, then treated with NTZ) and C9 (vaccinated, immunosuppressed, then infected).
Regarding the mean sera IgM level; vaccination after immunosuppression caused a significant increase (P <0.05) the mean IgM sera levels in groups C4 (immunosuppressed then vaccinated) and C6 (immunosuppressed, vaccinated, infected, then treated with NTZ) where reached a highly significant increase (P <0.01) in group C5 (immunosuppressed, vaccinated, then infected). On the other hand, there were significant reduction (P <0.05) between the mean IgM sera levels in C5 (immunosuppressed, vaccinated, then infected) & C6 (immunosuppressed, vaccinated, infected, then treated with NTZ) groups. Indicating that there was improvement in the immune status in these groups.
Vaccination before immunosuppression caused a highly statistically significant increase (P <0.01) in the mean IgM sera level in C8 (vaccinated then immunosuppressed) and C9 (vaccinated, immunosuppressed, then infected) groups. Statistically insignificant increase (P >0.05) was observed in group C10 (vaccinated, immunosuppressed, infected, then treated with NTZ). But there was significant reduction (P <0.05) between the mean IgM sera levels of C9 (vaccinated, immunosuppressed, and infected) & C10 groups (vaccinated, immunosuppressed, infected, and treated with NTZ) which may point to the presence of synergetic effect between NTZ treatment and the vaccine.
Regarding the mean sera IgA level, vaccination after immunosuppression caused a statistically insignificant reduction (P >0.05) in group C4 (immunosuppressed then vaccinated). A highly significant increase (P <0.01) in mean IgA sera level was seen in group C5 (immunosuppressed, vaccinated, then infected) and significant increased (P <0.05) in group C6 (immunosuppressed, vaccinated, infected, then treated with NTZ. Although, there were significant reduction (P <0.05) between the mean IgA sera levels of C5 (immunosuppressed, vaccinated, then infected) & C6 groups (immunosuppressed, vaccinated, infected, then treated with NTZ).
Vaccination before immunosuppression caused insignificant decrease (P >0.05) in the mean IgA sera level in group C8 (vaccinated then immunosuppressed). A highly statistically significant increase (P <0.01) was observed in the mean sera IgA levels in group C9 (vaccinated, immunosuppressed, then infected) and insignificant increase (P >0.05) in group C10 (vaccinated, immunosuppressed, infected, then treated with NTZ). But there was significant reduction (P <0.05) between the mean IgA sera levels of C9 (vaccinated, immunosuppressed, then infected) & C10 groups (vaccinated, immunosuppressed, infected, then treated with NTZ).
In conclusion group C10 (vaccinated, immunosuppressed, infected, then treated with NTZ) showed the highest improvement of the immune status which mean that NTZ could has a synergetic effect with the vaccination, and vaccination before immunosuppression better than vaccination after immunosuppression.
Histopathological examination of the intestinal tissue of the control infected groups (B1- Infected-untreated & C2- Immunosuppressed infected - untreated) revealed marked histopathological changes in the intestinal mucosa in comparison with the non-infected control groups (A- Uninfected – untreated & C1- Immunosuppressed). There were villous shortening and atrophy, goblet cell hyperplasia, marked inflammation and loss of brush border. Intestinal dysplasia was also detected.
Intestinal sections from mice received monotreatment with NTZ showed partial improvement in the pathological changes following Cryptosporidium infection.
In vaccinated groups (B3 & C7), (C4 – Immunosuppressed, then vaccinated) and (C8 – Vaccinated, then immunosuppressed), sections of the small intestine showed normal histological features in intestinal mucosa, however few inflamed villi were observed. Histopathological examination of intestinal sections in (B4- vaccinated then infected), (C5- Immunosuppressed, vaccinated, then infected) and (C9- Vaccinated, immunosuppressed, then infected) groups showed some histopathological changes in the intestinal mucosa as a result of infection with cryptosporidium parvum. These changes include mononuclear infiltrates in lamina propria, few blunt short broad villi, rarely seen villous epithelial hyperplasia, developmental stages of the parasite, and degenerations in some villous apical regions. Generally, both healthy and hypertrophied villi equally present in the same sections besides normal healthy cryptic profile, with some developmental stages of the parasite among the intervillous spaces.
Vaccinated, infected mice then treated with NTZ showed high improvement in the intestinal histopathological changes in immunocompetent and immunosuppressed groups (vaccinated before or after immunosuppression). Evidence of improvement included returning of the normal villous pattern and mild infiltration with inflammatory cells. The immunocompetent groups showed more improvement than immunosuppressed groups. Groups that were received vaccine before immunosuppression showed more normal intestinal structure than that received vaccine after immunosuppression.