Therapeutic efficacy of proton pump inhibitor (omeprazole) on cryptosporidiosis parvum in immunosuppressed experimental mice

Cryptosporidiosis is one of the most frequent food and water-borne diseases. The disease might be life-threatening in immunosuppressed patients. Unfortunately, the only approved drug, nitazoxanide, is with variable efficacies, particularly in malnourished children and immunocompromised patients. Therefore, there is a need to discover an alternative treatment that could be achieved by targeting the metabolic pathways. One of the important enzymes in the glycolysis pathway of C. parvum is triosephosphate isomerase, which could be hindered by the proton pump inhibitor (PPI) omeprazole. In this study, omeprazole was repurposed against C. parvum infection in experimentally immunosuppressed mice. This study was conducted on five mice groups (n = 10). Group I (Normal Control), group II (Infected Control): Mice were infected orally with 1 × 105C. parvum oocysts on the 15th day of DEX induced immunosuppression. Group III (NTZ-treated): infected and treated by NTZ. Group IV (Omeprazole-treated), and lastly, Group V (NTZ + Omeprazole-treated). The result obtained with omeprazole alone was better than nitazoxanide regarding oocyst shedding reduction percentages (84.9% & 56.1%, respectively). Also, it was better regarding restoration of histopathological and ultrastructural architectures, improvement of liver enzymes (alanine aminotransferase and aspartate aminotransferase) and renal functions (urea and creatinine), and the reduction of C. parvum triosephosphate isomerase (TIM) gene expression by RT-PCR. However, the best results were obtained with the combined treatment. Hence, omeprazole could be considered a novel drug option to treat this life-threatening parasitic infection either alone or combined with NTZ, especially in immunosuppressed patients.


Introduction
Cryptosporidium parvum is an apicomplexan protozoan parasite that can infect the microvilli of the gastrointestinal tract in a variety of vertebrates, including humans (Innes et al. 2020). It spreads by fecal-oral route through contaminated food or water with environmentally resistant oocysts (Efstratiou et al. 2017), and rarely by inhalation (Bauerfeind et al. 2016). Cryptosporidium is one of the most frequent causes of waterborne diseases (Pane and Putignani 2022). It comes second after rotavirus as a main worldwide cause of moderate-to-severe pediatric diarrhea (Kotloff et al. 2019). In the same context, it is considered the fourth leading cause of death among children under the age of five (GBD Diarrheal Diseases Collaborators 2017). Also, it was found accountable for 58% of the 936 worldwide reported waterborne outbreaks caused by protozoa between the years 1946 and 2016 (Efstratiou et al. 2017). Cryptosporidium can survive at high chlorine concentrations used in water treatment processes (Adeyemo et al. 2019); therefore, even swimming pools were involved as the source of infection in several outbreaks in Europe .
After ingestion of the infective oocysts, the released sporozoites invade the enterocytes of the small intestine (Love et al. 2017). The disease has an incubation period of 2-10 days, then it is usually presented either as self-limited gastroenteritis in immunocompetent humans or prolonged chronic illness in immunocompromised patients. Symptoms include watery diarrhea with abdominal pain, nausea, and vomiting; however, non-specific symptoms such as headaches, myalgia, fatigue, and low-grade fever might occur (Blanchard et al. 2015). In severe immunocompromised cases, extraintestinal manifestations involving the biliary tract, pancreas, joints, and respiratory system were also reported (López-Vélez 1995). Unfortunately, the disease may be life-threatening in those patients, as the severity, duration of symptoms, and response to treatment depend on the immune status of the host (Dhal et al. 2022).
The obstacle during the treatment of cryptosporidiosis is that the only approved drug by the (Food and Drug Administration) FDA, nitazoxanide, is with variable efficacies; particularly in malnourished children and immunocompromised patients (Checkley et al. 2015). Also, other available drugs against cryptosporidiosis such as azithromycin, paromomycin, and antiretroviral drugs (Gargala 2008) are not effective with apparent side effects, drug toxicity, and reports of resistance (Abdelmaksoud et al. 2020). Thus, owing to the limited treatment choices for this protozoal infection, there is an urgent need for the discovery of alternative therapies (Rahman et al. 2022).
Fortunately, targeting the metabolic pathways of parasites allows greater opportunities to select new drug treatments (Striepen and Kissinger 2004). In this situation, C. parvum relies mainly on anaerobic glucose oxidation for energy metabolism and the generation of ATP in the glycolytic pathways (Dhal et al. 2022). Nitazoxanide acts against C. parvum by hindering the anaerobic energy transfer reactions through inhibition of the parasitic enzyme pyruvateferredoxin oxidoreductase (Fox and Saravolatz 2005). One of the important enzymes in the glycolysis pathway of C. parvum is triosephosphate isomerase. This enzyme catalyzes the formation of glyceraldehyde 3-phosphate from dihydroxyacetone phosphate, which is a crucial third step that ensures the maximum ATP production for each glucose molecule (Nguyen et al. 2011).
Proton pump inhibitors (PPIs) belong to a group of benzimidazole-substituted compounds which are safely used to treat gastric and duodenal ulcers, and gastroesophageal reflux disease (García-Torres et al. 2016). Repurposing of the proton pump inhibitor omeprazole against G. lamblia showed cytotoxic effects with simultaneous inactivation of the triosephosphate isomerase enzyme (López-Velázquez et al. 2019). Moreover, PPIs showed antiparasitic effects against Leishmania donovani (Jiang et al. 2002), Trichomonas vaginalis (Shea et al. 2014), Schistosoma mansoni (Dina et al. 2019), and Entamoeba histolytica (Martínez-Pérez et al. 2020). Based on the previous literature, this study aims to evaluate the therapeutic efficacy of the PPI omeprazole compared to nitazoxanide against C. parvum infection in an experimentally immunosuppressed murine model.

Experimental animals
This study was conducted on 50 Swiss albino mice aged 6-8 weeks and weighing on average 20 ± 5 g. Throughout the study, mice were kept under controlled temperature and humidity conditions (25 °C; 70%). They were fed on a standard diet and water ad libitum and kept for 15 days adaptation period before inclusion in this trial. All experimental animal studies were conducted under international valid guidelines after approval by the Scientific Research Ethical Committee, Faculty of Medicine Menoufia University (IRB: 1/202/ PARA12).

Parasite and infective inoculum
Cryptosporidium parvum oocysts were obtained from the Parasitology department; Theodor Bilharz Research Institute preserved in 2.5% potassium dichromate (K 2 Cr 2 O 7 ) solution at 4 °C. The infective inoculum was prepared by washing the stored solution with distilled water and repeated centrifugation. Oocysts were concentrated by floatation in Sheather's sugar solution by centrifugation at 2000 r.p.m. for 10 min. The upper third of the solution in each tube was transferred into another tube. Oocysts were washed by centrifugation in distilled water 3 times, then suspended in phosphate-buffered saline (pH 7.2). The infective inoculum was adjusted to contain approximately 1 × 10 5 C. parvum oocysts/mouse (Asadpour et al. 2018).

Tested drugs preparation and dosing
Nitazoxanide Nitazoxanide (NTZ) (Nitazode 100 mg / 5 ml, Al Andalous Medical Company, Egypt) was given orally at a dose of 100 mg/kg/d starting on the 10th day p.i. and continued for 7 consecutive days (El-Wakil et al. 2021).

Omeprazole
Omeprazole (Omepral 20 mg capsules, manufactured by Memphis, Pharmaceuticals, Egypt) was dissolved and given orally at a dose of 20 μg/day starting on the 10th day p.i. and continued for 7 consecutive days (Tian et al. 2014).

Induction of immunosuppression
Immunosuppression was induced by giving the synthetic corticosteroid dexamethasone (DEX) (Dexazone 0.5 mg tablets, Kahira Pharmaceuticals and Chemical Industries Company, Cairo, Egypt) orally at a dose of 0.25 μg/kg/day for 14 successive days before inoculation with C. parvum oocysts. The immunosuppressed mice groups continued to receive dexamethasone at the same dose throughout the experiment (Rehg et al. 1988).

Study design
The experimental mice were divided into the following five groups (10 mice each): Group I (Normal Control): a group of healthy mice received saline only. Group II (Infected Control): Mice were infected orally with 1 × 10 5 C. parvum oocysts on the 15th day of DEX-induced immunosuppression. Group III (NTZ-treated): Mice were infected and treated by NTZ on the 10th d.p.i at a dose of 100 mg/kg body weight for 7 consecutive days. Group IV (Omeprazoletreated): Mice were infected and treated with omeprazole on the 10th d.p.i at a dose of 20 μg/d for 7 consecutive days. Lastly, Group V (NTZ + Omeprazole-treated): Mice were infected and treated by NTZ combined with omeprazole, as described in GIII and GIV.

Sampling
Fresh fecal pellets from each mouse in the infected groups were collected separately on the 11th, 14th, 17th, 21st, and 24th d.p.i. The mice were observed for their survival throughout the experiment. All the surviving mice were anesthetized by ether inhalation before being sacrificed by cervical dislocation on the 24th p.i.d. Blood samples were collected, and sera were separated by centrifugation and stored at − 20 °C for further biochemical studies. From each mouse, the terminal two cm of the ileum and samples from lung tissues were taken and fixed in 10% neutral buffered formalin for histopathological examination. A second part of the ileum was fixed in 5% glutaraldehyde for transmission electron microscopic study (TEM), and a third part of the ileum was immediately stored at − 80 °C for the molecular study (RT-PCR).
Evaluating the efficacy of treatment 1. Oocyst shedding Each collected fecal sample was weighted, dissolved in 10% formalin, passed through sterile gauze, and 50 μl was taken and put on glass slides, then stained with the modified Ziehl-Neelsen stain (cold method). The stained slides were examined under the oil immersion lens (× 100) to determine the number of oocysts (Garcia 2007). The oocysts number for each mouse was determined by counting oocysts in ten microscopic fields, and the arithmetic mean was calculated to detect the number of oocysts/ grams of feces. The percentage of reduction in oocyst shedding was calculated according to the following equation (Moawad et al. 2021).

Histopathological study
For each mouse, the preserved ileum and lung tissues in buffered formalin were dehydrated in ascending grades of ethanol, followed by immersion in xylene then impregnated in paraffin blocks, and finally, serial sections of 5 μm in thickness were prepared and stained with hematoxylin and eosin (H & E) (Drury and Wallington 1980).

Transmission electron microscopy (TEM)
Intestinal tissue specimens were prepared for Transmission Electron Microscopy (TEM) using the procedures described previously (Winey et al. 2014  (Gene ID: 3371710) were forward 5ˋ-GGG TTT GGA GAA TGG CCT GA-3ˋ and reverse 5ˋ-GCC CAA ATT GGC TCG TAA GC-3ˋ. And GAPDH gene was used as an endogenous control (forward 5ˋ-ACC ACA GTC CAT GCC ATC AC-3ˋ and reverse 5ˋ-TCC ACC ACC CTG TTG CTG TA-3ˋ). The real-time cycler was programmed according to the following conditions: PCR initial activation step for one minute at 95 °C followed by three-step cycling of denaturation for 30 s at 95 °C, annealing for 30 s at 60 °C, and final extension for 10 min at 72 °C. Melting curve analysis was performed to verify the specificity and the identity of the PCR products using 7500 software version 2.0.1.

5.
Biochemical studies for alanine aminotransferase, aspartate aminotransferase, urea, and creatinine Alanine aminotransferase (ALT) (MAK052, Sigma-Aldrich, St. Louis, USA) and Aspartate aminotransferase (AST) (MAK055, Sigma-Aldrich, St. Louis, USA) enzyme activities were measured in serum samples according to kits protocols. The reactions were read by a fully automated chemistry autoanalyzer (AU 680, Beckman Coulter, California, USA) at 530-550 nm and the activities were expressed as U/L. Moreover, serum urea (ab83362, Abcam, USA) and creatinine (ab65340, Abcam, USA) were measured to evaluate renal functions, and the reactions were expressed as mg/ dl.

Statistical analysis
Data were collected, tabulated, and statistically analyzed using an IBM-compatible personal computer with Statistical Package for the Social Sciences (SPSS) version 26. Quantitative data were expressed as mean (x̅ ), standard deviation (SD), and One-way ANOVA (F) test was utilized to compare between over two groups having normally distributed variables followed by Least Significant Difference (LSD) as a post hoc test. Spearman correlation was used to investigate the relationship between oocyst shedding and biochemical changes. Probability of error (P-value) P > 0.05 was not significant, P < 0.05 was significant, and P < 0.001 was highly significant.

Results
In the current study, tow mice from the infected control group (GII) died; therefore 8 mice are included in the results of this group (n = 8). While, in all treated groups the survival rate was 100%.

Oocyst shedding
Regarding oocyst shedding ( Fig. 1), in the infected control group (GII) the number of oocysts/g of feces × 10 3 increased gradually and reached its peak (129.5 ± 3.3) on the 21st p.i.d. then it declined. Oocyst shedding in all treated groups (GIII, GIV & GV) showed statistically significant differences (P < 0.001) when compared to GII.
Omeprazole combined with nitazoxanide showed the best results, followed by omeprazole and lastly nitazoxanide (RP% were 92.3, 84.9, and 56.1, respectively, at the end of the experiment). Omeprazole-treated group (GIV) started on the 14th p.i.d. and continued to the 24th day to show significant differences when compared to the NTZ-treated group (GIII). While the Omeprazole + NTZ-treated group (GV) showed significant differences (P<0.001) when compared to GIV and GIII throughout the experiment (Table 1).

Ileal tissue
Histopathological examination of ileum samples obtained from the normal control group (GI) showed preserved cryptvillous ratio with long villi, intact epithelial lining, regular brush border, and normal numbers of goblet cells (Fig. 2a, b). The infected control group (GII) showed disturbed villous architecture with blunted villous tips and broadening of villi, ulceration, and focal sloughing of villous tips in the lumen, focal or total loss of the brush border, and many C. parvum oocysts were found embedded in villous epithelium together with inflammatory cells infiltration in lamina propria and congestion (Fig. 2c, d).
NTZ-treated group (GIII) showed some sort of improvement in surface epithelium with partial restoration of crypt villous ratio and regular brush border. However, focal blunting, thickening of some villi, inflammation and congestion were still present (Fig. 3a). OMP-treated group (GIV)  1 3 showed obvious restoration of villous architecture, regular brush border with goblet cells, absence of C. parvum oocyst, and minimal inflammation (Fig. 3b, c). While the OMP + NTZ-treated group (GV) showed marked improvement when compared to GIII and GIV. There was a restoration of villous architecture, brush border, goblet cells, minimal inflammation, and absence of the oocysts (Fig. 3d).

Lung tissue
Histopathological sections of lung tissues from the normal control group (GI) showed normal alveoli and bronchi without pulmonary hemorrhage or interstitial inflammation or oocysts at the epithelial lining (Fig. 4a). the infected control group (GII) showed obvious destruction of intra-alveolar septa, pulmonary hemorrhage, focal ulceration, inflammation of the bronchial lamina propria with many cryptosporidium oocysts at the epithelial lining of bronchi (Fig. 4b, c). NTZ-treated group (GIII) showed interstitial inflammation, bronchial inflammation, and pulmonary hemorrhage, but no oocyst at the epithelial lining was detected (Fig. 4d). OMP-treated group (GIV) showed marked improvement with normal epithelial lining of bronchi, mild interstitial inflammation, mild hemorrhage, and no oocysts were found (Fig. 4e). The OMP + NTZ-treated group (GV) showed normal epithelial lining without oocysts, minimal interstitial inflammation, and minimal pulmonary hemorrhage (Fig. 4f).

Transmission electron microscopy (TEM)
Transmission electron microscopic study of the ileum tissues obtained from the normal control group (GI) showed normal columnar epithelium, nuclear chromatin, well-developed apical microvilli, mucin-secreting goblet cells, mitochondria of normal shape and number, and intact intercellular junction (Fig. 5a, b). Infected-control group (GII) showed discontinuity, destroyed intercellular junctions, loss of microvilli, shrunken and deformed pyknotic nuclei with a lot of nuclear heterochromatin, fragmented or swollen mitochondria with dilated cristae, and multiple C. parvum oocysts were detected at the site of severely damaged microvilli (Fig. 5c-f).
NTZ-treated group (GIII) showed partial regeneration of columnar epithelium with prominent microvilli; however, abnormal mitochondria, damaged intercellular junction, pyknotic nuclei, goblet cells with decreased mucin secretion, and cytoplasmic vacuoles were still present (Fig. 6a, b). The OMP-treated group (GIV) showed regenerated columnar epithelium with prominent microvilli, normal nuclei, and intact intercellular junction but occasionally there were fewer mitochondria or fragmentation, dilated Golgi bodies, cytoplasmic vacuoles, and multiple vacuolated C. parvum oocysts were found (Fig. 6c-f). OMP + NTZ-treated group (GV) showed obvious regeneration of the columnar epithelium with prominent microvilli, normal nuclei, mitochondria of normal shape and number, intact intercellular junctions, and no C. parvum oocyst was seen (Fig. 6g, h).
3. Real-time PCR for C. parvum triosephosphate isomerase gene expression In the present study, RT-PCR for C. parvum triosephosphate isomerase gene expression fold changes in ileal tissues obtained from all treated groups (GIII, GIV, and GV) were statistically significant (P < 0.001) when compared to the infected control group (GII). There were statistically significant (P < 0.001) differences between OMP-treated (GIV) and OMP + NTZ-treated (GV) when compared to the NTZ-treated group (GIII). However, the difference between GIV and GV was not significant (P = 0.48) ( Table 2).
4. Biochemical studies for ALT, AST, urea, and creatinine . a Negative control group shows preserved alveoli and bronchi with no pulmonary hemorrhage or interstitial inflammation. The lining of the bronchi is normal and with no oocyst at the epithelial lining. b Positive control group shows marked destruction of intra-alveolar septa (black arrow), marked pulmonary hemorrhage) (red arrow) c Positive control group shows many Cryptosporidium oocysts at the epithelial lining of the bronchi (black circles), marked pulmonary hemorrhage (yellow star), and focal ulceration (green circle). d NTZ-treated group shows interstitial inflammation (green arrow), and inflammation in lamina propria of bronchi (black arrow) with no oocyst at the epithelial lining but there is marked pulmonary hemorrhage (yellow star). e The OMP-treated group shows minimal interstitial inflammation (black arrow), and minimal pulmonary hemorrhage (yellow star). The bronchi show normal epithelial lining without oocysts or hyperplasia. f OMP + NTZ-treated group shows marked improvement with mild interstitial inflammation and mild hemorrhage. The epithelial lining of the bronchi is normal without hyperplasia or oocysts, but mild inflammation is present in the lamina propria of the bronchi

Liver enzymes
Regarding alanine aspartate aminotransferase results, the infected control group (GII) showed a significant increase (74.4 ± 1.6) when compared to the normal control group (39.5 ± 2.01) (P < 0.001). NTZ-treated group (GIII) showed a significant decrease (44.4 ± 1.9) when compared to the infected control group (GII). However, this decline of ALT value in GIII did not reach the normal value of GI, so the difference between both groups was significant (P < 0.001). Both OMP-treated and OMP + NTZ-treated groups (GIV and GV) showed significant decreases of ALT values reaching normal values. Accordingly, the differences were significant (P < 0.001) when compared to GII, while on comparing both groups to GI and to each other, the differences were statistically not significant (P > 0.05) ( Table 2).
Regarding aspartate aminotransferase, the infected control group (GII) showed a significant increase (58.5 ± 2.6) when compared to the normal control group (30.8 ± 2.3) (P < 0.001). NTZ-treated group (GIII) showed a significant decrease (37.9 ± 1.5) when compared to GII. However, this decline of AST value in GIII did not reach the normal value of GI and the difference between both groups was significant (P < 0.001). The OMP-treated group (GIV) showed a significant decrease in the AST value (33.2 ± 1.8) when compared to GII and GIII. As the value was still above normal, Normal control group showing normal columnar epithelium with normal nuclear chromatin (N), well-developed apical microvilli (MV), mucin secreting goblet cell (GC), mitochondria (green arrow), intercellular junction (IJ) (blue arrow), and rough endoplasmic reticulum (RER). c Infected-control group is showing discontinuity, and loss of microvilli (red arrow) severely fragmented mitochondria (green arrow), and multiple vacuoles (V). d Infected-control group shows severely damaged microvilli (red arrows), edema, and destroyed intercellular junctions. e Infected control shows areas of severely damaged epithelium with total loss of microvilli (black arrow), cellular contents spilled into the lumen (yellow arrow), and multiple Cryptosporidium oocysts were present (red arrows). f The infected control group shows shrunken and deformed pyknotic nuclei (PN) with a lot of nuclear heterochromatin (N.H), swollen mitochondria with dilated mitochondrial cristae (red arrows), and destroyed intercellular junction the difference was significant (P < 0.001) when compared to GI. While the OMP + NTZ-treated group (GV) showed the return of AST value to the normal, thus the difference between GV and GI was not significant (P = 0.39), but comparing GV to GII, GIII, and GIV were highly significant (P < 0.001) ( Table 2).

Renal functions
Regarding serum urea results, the infected control group (GII) showed a significant increase (43.1 ± 1.96) when compared to the normal control group (29.0 ± 2.4) (P < 0.001). NTZ-treated group (GIII) showed a significant Fig. 6 Photomicrographs of TEM of the ileum from (a, b) NTZ-treated group shows partial regenerated columnar epithelium with prominent microvilli (MV), cytoplasmic vacuoles and edema still present (V), abnormal mitochondria, less prominent intercellular junction, a pyknotic nucleus with excess heterochromatin (N), and goblet cell (GC) with decreased mucin secretion. c OMP-treated group shows regenerated columnar epithelium with prominent microvilli (MV), normal nucleus (N) with normal euchromatin, and preserved intercellular junction (ICJ) but there is a decreased number of mitochondria. d OMP-treated group shows partially regenerated columnar epithelium with prominent microvilli (MV), restored goblet cell number (GC), and preserved intercellular junction (ICJ) mitochondria that were of normal number but still deformed in shape (fragmented) (red arrow), and cells show cytoplasmic vacuoles. e OMPtreated group shows regenerated columnar epithelium with prominent microvilli (MV). Mitochondria are of normal number (green arrow), but edema and multiple cytoplasmic vacuoles (V) were still present. f OMPtreated group shows regenerated columnar epithelium with prominent microvilli (MV), multiple vacuolated oocysts (red arrows), and dilated Golgi (G). g, h OMP + NTZ-treated group shows euchromatic nuclei (N), nucleolus (Nu), prominent microvilli (MV), normal mitochondria shape and number (green arrows), normal intercellular junctions (ICJ), and no Cryptosporidium oocysts in the epithelium decrease (33.0 ± 1.8) when compared to the infected control group (GII). However, this decline of serum urea in GIII did not reach the normal value of GI; thus, the difference between both groups was significant (P < 0.001). Both OMP-treated and OMP + NTZ-treated groups (GIV and GV) showed a significant decrease in serum urea values reaching the normal value. Accordingly, the differences were significant (P < 0.001) when compared to GII, while comparing both groups to GI was not significant (P > 0.05). The best result was obtained in the OMP + NTZtreated group (GV) which showed highly significant differences when compared to GIII and GIV (Table 2).
Concerning serum creatinine results, the infected control group (GII) showed a highly significant increase (3.32 ± 0.1) when compared to the normal control group (GI) (0.96 ± 0.01) (P < 0.001). NTZ-treated group (GIII) showed a significant decrease (1.7 ± 0.05) when compared to the infected control group (GII). However, serum creatinine in GIII was still significantly higher than in the normal control group (GI) (P < 0.001). Both OMP-treated and OMP + NTZ-treated groups (GIV and GV) showed significant decreases in serum creatinine, but they did not reach the normal value. Accordingly, the differences were significant (P < 0.001) when compared to GII and GIII. The differences were statistically not significant (P > 0.05) when both groups were compared to GI and to each other ( Table 2). The results obtained in the OMP + NTZ-treated group (GV) were highly significant when compared to GIV.

Spearman correlation between oocyst shedding and biochemical changes
Spearman correlation test revealed positively significant (P < 0.001) relationships between oocyst shedding numbers and the measured biochemical parameters. The correlation coefficients (r) of ALT, AST, serum urea, and serum creatinine were 0.857, 0.932, 0.843, and 0.951, respectively (Fig. 7).

Discussion
Results of the current study revealed that oocyst shedding in the infected control group increased gradually and reached its peak on the 21st p.i.d. then it declined. The reduction percentages in all treated groups were statistically significant when compared to the infected control group. Moreover, omeprazole combined with nitazoxanide showed the best therapeutic result (92.3%) followed by omeprazole (84.9%) and lastly nitazoxanide (56.1%).
In the current study, nitazoxanide results agree with Shoultz et al. (2016) who found that nitazoxanide has a cure rate of only 56% in undernourished children. Similarly, Taha et al. (2017) stated a 52.7% reduction percentage of Cryptosporidium oocyst shedding on the 21st day post-infection after nitazoxanide treatment. Also, El-Hawary et al. (2022) reported an oocyst shedding reduction percentage of 54%. The therapeutic efficacy of NTZ in the present study was higher than the results documented by Fahmy et al. (2020) and Esmat et al. (2022) who reported 39% and 38%, respectively. On the contrary, the results in this study were lower than those obtained by El-Wakil et al. (2021) and Farid et al. (2022) who reported 66% and 92.05%, respectively. This controversy could be attributed to giving NTZ at a different dose and/or duration. For instance, Farid et al. (2022) in their study, gave NTZ at a dose of 100 mg/kg/d continuously for fourteen days; while in this study, NTZ was given at the same dose but for seven days.
In the present study, RT-PCR for C. parvum triosephosphate isomerase gene expression fold change in ileum tissues obtained from all treated groups was statistically significant when compared to the infected control group. Omeprazole alone or combined with NTZ was statistically significant when compared to nitazoxanide alone. The assessment of parasite load by qRT-PCR has an advantage when compared with qPCR as RNA used in the former, due to its quick degradation is considered a better indicator of cell viability than DNA (Zhang and Zhu 2020). Accordingly, the significant reduction of triosephosphate isomerase gene expression which indicates reduced oocyst viability together with reduced oocyst shedding obtained with omeprazole alone or combined with NTZ could be attributed to the successful targeting of this crucial gene in C. parvum glycolysis pathway by omeprazole and the occurrence of a synergistic action between both drugs in the combined-treated group (GV). To the best of our knowledge, this is the first study utilizing omeprazole against cryptosporidiosis in experimental animals. This significant therapeutic efficacy of omeprazole is in accordance with García-Torres et al. (2016) and López-Velázquez et al. (2019) who stated the effectiveness of omeprazole against G. lamblia and referred this efficacy to the direct inhibition of triosephosphate isomerase enzyme.
It was reported that C. parvum alters the gut microbiota of infected mice (Mammeri et al. 2019). Recently, Rahman et al. (2022) showed that the abundance of Helicobacter pylori positively correlates with the severity of C. parvum infection or oocyst shedding. Therefore, we suggest that the therapeutic agent having an influence on gut microbiota may have indirect antiparasitic effect. Interestingly, omeprazole besides its direct action on TIM, it affects gut microbiota either directly by targeting the proton pumps of the bacteria and fungi, or indirectly through affecting the microenvironment of the flora through changing the pH (Vesper et al. 2009). Furthermore, administration of omeprazole with nitazoxanide in combination with other antibiotics was found successful in treatment of H. pylori (Basu et al. 2011). Histopathological examination and the ultrastructural study of ileum tissues obtained from the normal control group showed preserved crypt-villous ratio, intact epithelial lining, regular brush border, normal secretory goblet cells, and intact intercellular junctions. While the infected control group showed disturbed villous architecture, ulcerations, inflammatory infiltration in lamina propria, congestion, dysplasia, decreased numbers of goblet cells, focal or total loss of the brush border microvilli, and destroyed intercellular junctions. These pathological alterations induced by C. parvum were in accordance with those observed in several previous studies (Hassan et al. 2021(Hassan et al. , 2022Moawad et al. 2021). Also, it was stated that the columnar epithelial cells and goblet cells decrease in the ileum, reducing the crypt-villous ratio at the time when the number of C. parvum oocysts is the highest (Sasahara et al. 2003). Regarding the ultrastructural findings in both normal and infected control groups, similar observations were reported by Al-Mathal and Alsalem, (2013) and Esmat et al. (2022).
In the current work, treatment with NTZ caused some sort of histopathological improvement with the persistence of C. parvum oocysts, blunting and thickening of some villi, and inflammation. The ultrastructural study also confirmed this partial regeneration of the columnar epithelium. Omeprazole treatment caused an obvious histopathological improvement, restoration of villous architecture, minimal inflammation, normal brush border, and goblet cells. The TEM study revealed regeneration of columnar epithelium with prominent microvilli, normal nuclei, and preserved intercellular junction. The best histopathological and ultrastructural improvements were obtained with the combined OMP + NTZ treatment, which resulted in the complete restoration of villous architecture. These improvements correlate with the oocyst shedding reduction rates obtained with nitazoxanide, omeprazole and omeprazole combined with nitazoxanide (56.1%, 84.9%, and 92.3%, respectively).
In harmony with the current work results, several previous studies reported better therapeutic efficacies of the new drug candidates when they were compared to NTZ in immunosuppressed mice models. For example, El-Wakil et al. (2021) reported a significantly higher therapeutic effect of mefloquine than NTZ on experimental cryptosporidiosis, and 100% oocyst clearance with the restoration of normal epithelial architecture was obtained in the combined group. They explained the partial improvement with NTZ as the impaired immune response decreases the therapeutic efficiency of this drug (Atia et al. 2016;Dhal et al. 2022). Also, Hassan et al. (2022) documented that the treatment with NTZ loaded with silver nanoparticles showed the highest oocyst shedding reduction, with significant improvement in the histopathological changes when compared to NTZ alone. Furthermore, Esmat et al. (2022) stated that NTZ monotherapy showed the least efficacy, and it failed to restore normal intestinal architecture. While the combined clofazimine and NTZ treatment resulted in significant improvements in the parasitological, histopathological, and ultrastructural findings.
In a previous study, Certad et al. (2010) reported respiratory cryptosporidiosis with respiratory failure in immunosuppressed patients. Thus, in the present study, a histopathological examination of lung tissues was done to evaluate the tested drug's therapeutic efficacy. The normal control group showed intact alveoli and bronchi. While, the infected control group showed obvious destruction of intra-alveolar septa, pulmonary hemorrhage, focal ulceration, and inflammation of the bronchial lamina propria with many Cryptosporidium oocysts at the epithelial lining of the bronchi.
Treatment with nitazoxanide resulted in partial improvement, while omeprazole therapy caused an obvious restoration of the normal epithelial linings with mild interstitial inflammation and hemorrhage. Also, the best results were obtained with the combined NTZ + OMP treatment. Taha et al. (2017) reported similar results and improvement of lung pathology in the infected immunosuppressed mice after the combined treatment with NTZ and atorvastatin. Also, Moawad et al. (2021) reported similar results in their study on NTZ loaded with chitosan nanoparticles against experimental cryptosporidiosis in immunosuppressed mice.
Regarding ALT and AST results in this study, the infected control group showed a significant increase when compared to the normal control group. NTZ treatment caused significant decreases in ALT and AST when compared to the values of the infected control group. However, this decline did not reach the normal values. On the other side, OMPtreated and OMP + NTZ-treated groups showed significant decreases when compared to infected control and nitazoxanide-treated groups. The combined treatment reverted the elevated liver enzymes to normal values.
In the present study, the significantly increased serum levels of ALT and AST indicates the occurrence of C. parvuminduced hepatocyte damage as serum ALT indicates cell membrane injury while AST denotes mitochondrial damage in hepatic tissues (AbouGabal et al. 2015). These findings are in accordance with Aboelsoued et al. (2019) and Elmahallawy et al. (2020) who recorded elevations of ALT and AST in experimentally infected mice with Cryptosporidium. Which could indicate the extra-intestinal harmful effect of cryptosporidiosis (Chalmers and Davies 2010). In the current study, the Spearman correlation test revealed positively significant relationships between oocyst shedding and serum ALT and AST values. Thus, the restorations of normal values of liver enzymes were correlated to the therapeutic efficacy and oocyst-shedding reduction rates of the tested drugs.
The results of serum urea and creatinine in this study showed highly significant increases in the infected control when compared to the normal control group. The NTZ-treated group showed significant decreases when compared to the infected control group, but they were still significantly higher than the normal values. Both omeprazole and the combined treatment showed significant decreases in serum urea and creatinine values, but creatinine did not reach normal levels. However, the results obtained with the combined treatment were the best. Also, the Spearman correlation test revealed positively significant relationships between oocyst shedding and serum urea and creatinine.
Concerning this obvious renal impairment during this experiment, which correlates with oocyst shedding rates, it is suggested that severe watery diarrhea in immunosuppressed mice might be involved. One of the mechanisms which are thought to contribute to the pathogenesis of Cryptosporidium-induced diarrhea is the impaired intestinal epithelial barrier function and increased permeability (Ott et al. 1991). Disruption of this barrier integrity and function via altering the expression of tight junction (TJs) and adherens junction (AJs) proteins by C. parvum infection were studied. The defects in these barriers have been related to the onset of diarrhea, inflammation, and other extra-intestinal organ affection (Kumar et al. 2018). Interestingly, in the current TEM study, intraepithelial junction destruction was observed in the infected control group with complete restoration after the successful combined treatment.
In the same context, hospitalized diarrheal patients might be complicated with acute kidney injury (AKI) which occurs primarily due to extracellular volume losses from the gastrointestinal tract. These volume losses could be linked to the damaged intestinal TJs which are essential for the maintenance of the electrochemical gradients and transcellular ion transport (Viswanathan et al. 2009). The risk for the development of this renal injury was found higher among HIV/AIDS patients (Bradshaw et al. 2018) which may be attributed to the prolonged course of the disease and the concomitant failure of treatment. Recently, Agnew et al. (2021) reported a case of a postinfectious inflammatory syndrome associated with AKI after C. parvum infection in an immunosuppressed patient. Therefore, renal functions should not be neglected during the follow-up of severe cryptosporidiosis cases, especially in immunocompromised patients.

Conclusion
This study is the first to repurpose the PPI omeprazole against C. parvum infection in experimentally immunosuppressed mice. Despite omeprazole is not approved as antiparasitic agent, it was better than the only FDA-approved drug nitazoxanide regarding oocyst shedding reduction percentage, restoration of histopathological and ultrastructural architectures, improvement of liver enzymes and renal functions, and the reduction of C. parvum triosephosphate isomerase gene expression. However, the best results were obtained with the combined treatment. Thus, further in vitro, experimental, and clinical studies are required to evaluate omeprazole as a novel drug option to treat this life-threatening parasitic infection either alone or combined with NTZ, especially in immunosuppressed individuals.
Author contributions All authors prepared the research protocol and the study design. MM Saleh and FH Shalaan carried out the experimental infection in mice, administrated the therapeutic doses, follow up of the mice, the parasitological study and tissue sampling for biochemical and molecular studies. MA Kora examined the stained ileum and lung sections then interpreted the histopathological results. MM Saleh and EVN Beshay interpreted the transmission electron microscopy results. MM Saleh collected and tabulated the data while EVN Beshay and FH Shalaan participated in analysis and interpretation of the data. EVN Beshay prepared and wrote the manuscript. NE Nassef and O El Shafey critically revised the manuscript. All authors had read and approved the final manuscript version.
Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.