Assessment of chitosan nanoparticles in improving the efficacy of nitazoxanide on cryptosporidiosis in immunosuppressed and immunocompetent murine models

Abstract

Cryptosporidiosis is one of the major causes of diarrhea in immunocompetent and immunocompromised patients. It is self-limited in immunocompetent individuals. However, in the immunocompromised it can cause life-threatening diarrhea and results in chronic malabsorption of fluids, vitamins and electrolytes resulting in wasting. Our study is concerned with assessing and comparing the efficacy of nitazoxanide (NTZ) alone and NTZ loaded chitosan nanoparticles (NTZ loaded CS NPs) in the treatment of experimental cryptosporidiosis using parasitological and histopathological parameters. One hundred mice were divided into 5 groups (20 mice each). Each group was divided into 2 subgroups according to the immune status [a-immunocompetent, b-immunosuppressed]. group 1: control (healthy), group 2: control infected by Cryptosporidium oocysts, group 3: infected treated by NTZ, group 4: infected then treated by NTZ loaded CS NPs and group 5: infected then treated by chitosan nanoparticles (CS NPs) alone. Treatment of Cryptosporidium infected mice with NTZ loaded on CS NPs resulted in the highest significant reduction in oocysts shedding in both immunocompetent and immunosuppressed groups followed by treatment with NTZ form then by treatment with CS NPs alone. The treatment with NTZ loaded CS NPs displayed a remarkable improvement of the histopathological changes of the intestine, liver and lung while NTZ treated group showed some improvement. Treatment with NTZ loaded CS NPs in murine cryptosporidiosis gave the best results as it caused marked reduction in fecal oocysts counts and improvement of histopathological changes in immunocompetent and immunosuppressed groups.

Introduction

Cryptosporidiosis is a common zoonotic parasitic disease caused by various Cryptosporidium species. It has a worldwide distribution. The highest prevalence has been detected in developing countries with high mortality in children below age of 2 years. Generally, the disease produces watery diarrhea and anorexia in susceptible hosts (Chalmers and Katzer 2013; Bouzid et al. 2018). Infection results from ingestion of oocysts which are excreted by infected people or animals. It is transmitted via consumption of contaminated food or water. Development of this parasite is relatively restricted to the intestine but the biliary tract, pancreas and lungs may be also affected in immunosuppressed host (McCole et al. 2000).

Cryptosporidium species cause several pathological changes in the epithelial cells lining the gastrointestinal tract. It leads to loss of the intestinal microvillus border, shortening of the villi and lengthening of crypts, submucosal oedema and mononuclear cells inflammatory infiltration in the lamina propria causing acute gastrointestinal disturbance (Enemark et al. 2003; Fayer and Xiao 2008).

Although the fact that Cryptosporidium infection has been identified as a significant cause of diarrheal illness for more than three decades, specific treatment is limited (Cabada and White 2010). Food and Drug Administration (FDA) approved that nitazoxanide (NTZ) is the drug of choice in treatment of this pathogen (Checkley et al. 2015). NTZ was synthesized in 1980s by combining a thiazole ring with a benzamidine ring. It is a broad-spectrum anti-parasitic drug which is used as an anti-helminthic and anti-protozoal drug (Cabada and White 2010). It acts through inhibition of pyruvate ferredoxin oxidoreductase (PFOR), an essential enzyme in the anaerobic energy metabolism (Hoffman et al. 2007). While NTZ was an important advance in cryptosporidiosis treatment in children, it has limited efficacy in immunocompromised and malnourished individuals, which raised the importance for development of better drugs for cryptosporidiosis therapy (Checkley et al. 2015).

Over the last few years, nanoparticles (NPs) have gained great attention in the field of drug delivery and treatment (De Jong and Borm 2008).The development of these NPs into smart systems for coating of therapeutic agents allows drug delivery to the target sites with controlled release. Moreover, this targeted delivery reduces the drug side effects and improves patient’s compliance with reduced dosing frequency (Rizvi and Saleh 2018).

CS is a very important chitin derivative which is made by extensive chitin deacetylation. It is obtained from crustacean shells, also from the cell walls of fungi and cuticle of insects. It is a naturally occurring cationic polysaccharide, mucoadhesive biocompatible polymer and approved by the U.S. FDA for drug delivery (Varum et al. 1994; Sorlier et al. 2001).

In cryptosporidiosis, CS conjugated with polyvinyl alcohol was confirmed to inhibit the connection of sporozoites of Cryptosporidium to enterocytes in vitro (Luzardo Álvarez et al. 2012). A study by Ahmed et al (2019) elucidates CS NPs as an effective anti-cryptosporidial agent. The study was performed in vitro and confirmed in mice in vivo infectivity assay.

In this work, the efficacy of NTZ loaded CS NPs was assessed by parasitological and histopathological examinations in murine model.

Materials and methods

Animals

The study was conducted on one hundred male Swiss albino mice, about 4–6 weeks in age and 20–25 g in weight. Biological Supply Center at Theodor Bilharz Research Institute (TBRI) was the source of obtaining the mice. They were preserved on a typical diet with permitted available water in an animal house at 20–22 °C. There was no intestinal parasitic infection in the mice, which was confirmed by feces examination on three days consecutively using formol–ether concentration technique (Ridley and Hawgood 1956) and modified Ziehl–Neelsen stain (Henriksen and Pohlenz 1981).

Experimental design

Mice were divided into 5 groups (20 mice each). Each group was subdivided into 2 subgroups (subgroup a and b) according to immune status [immunocompetent and immunosuppressed respectively]: group 1: healthy control, group 2: control infected by Cryptosporidium oocysts, group 3: infected then treated by NTZ, group 4: infected then treated by NTZ loaded CS NPs, and group 5: infected then treated by CS NPs only.

Immunosuppression

Immunosuppression was induced in mice by giving dexamethasone (Dexazone 0.5 mg tablets) orally, using esophageal tube at a dosage of 0.25 µg/gm/day continued for 14 days successively previous to infection with Cryptosporidium oocysts. The mice were maintained on the same dose of dexamethasone during the experiment (Rehg et al. 1988). Dexazone (0.5 mg) was manufactured and provided by Kahira Pharmaceuticals and Chemical Industries Company, Shoubra-Cairo, Egypt. Then the murine clinical picture was assessed.

The infection

Cryptosporidium parvum oocysts were obtained from Parasitology department; Theodor Bilharz Research Institute. The samples were proven to be positive for Cryptosporidium by modified Ziehl–Neelsen staining method. Oocysts of Cryptosporidium parvum were conserved by mixing with an equal amount of 2.5% potassium dichromate (K2Cr2O7) and kept at (4 °C) till use for infection (Current et al. 1983; Campbell and Current 1983; Khalifa et al. 2001). The infective inoculum was prepared in accordance with Reese et al. (1982). For counting Cryptosporidium spp. oocysts in 1 ml of the infective inoculum, three slides were prepared from the sample; each of them consisted of 50 μl modified Zeihl–Neelsen stained specimen and oocysts were counted to calculate the mean number of oocysts then multiplied by 20 to obtain their concentration in 1 ml of the sample (Garcia and Bruckner 1997). All mice except the healthy control groups were orally infected with Cryptosporidium oocysts using esophageal tube. Each mouse was infected with about 10⁴ oocysts (Gaafar 2007). The immunosuppressed groups were inoculated with Cryptosporidium oocysts on the 15th day of dexamethasone therapy (Reese et al. 1982; Moon et al. 1982). To ensure that the mice had been infected, fecal samples were collected from infected mice with Cryptosporidium from the second day post infection to the 7th day post infection and examined by Modified Zeihl–Neelsen stain to detect Cryptosporidium oocysts (Garcia 2007).

Regimen of treatment

NTZ (Nanazoxid) was obtained from Medizen Pharmaceutical Industries for Utopia Pharmaceuticals. It was given to group (3) at a dose of 200 mg/kg/day (Li et al. 2003). NTZ loaded CS NPs was given to group (4) in a dosage of 200 mg/kg/day, while mice of group (5) received 20 µg of CS NPs in 200 µl of PBS/mouse/day. These drug formulations were given to mice seven days post infection and continued for 3 days in immunocompetent and 6 days in immunosuppressed to sacrifice mice after three weeks post-infection by rapid decapitation.

Preparation of CS NPs

CS; 93% degree of deacetylation,sodium tripolyphosphate (TPP), acetic acid and phosphate buffer saline (PBS) were obtained from Sigma-Aldrich, USA.

CS NPs were manufactured by the ionotropic gelation of CS with tripolyphosphate (TPP) anions. CS was liquefied in acetic aqueous solution. The ratio of CS concentration to acetic acid in aqueous solution was adjusted to be 1:1.5. The TPP solution was made by double-distilled water at concentration (1 mg/ml). CS NPs were prepared by the gradual addition of 5 ml of the CS solution on 2 ml of TPP solution. An opalescent suspension was formed spontaneously by magnetic stirring at 1000 rpm for 1 h at room temperature. Separations of CS NPs were done by centrifugation at 20,000 ×g at 14 °C for 30 min, and then they were freezed-dried and stored at 4 °C. Their weights were calculated (Somnuk et al., 2011).

NTZ loaded CS NPs

NTZ loaded CS NPs were made by the dropwise adding of solution of CS to an aqueous solution of sodium TPP (having NTZ at concentration of 100 mg/ml) with constant stirring, followed by sonication. The separation of NTZ loaded NPs from aqueous suspension was done by centrifugation at 20,000 ×g for 30 min at 14 °C.

The encapsulation efficiency (AE) and loading capacity (LC) of NPs were calculated as follow:

$$\%\text{AE}=\left[\left(\text{A}-\text{B}\right)/\text{A}\right]\times 100$$

$$\%\text{LC}=\left[\left(\text{A}-\text{B}\right)/\text{C}\right]\times 100$$

where A is the total amount of NTZ, B is the free amount of NTZ and C is the weight of NPs.

Characterization

Scanning electron microscope (SEM) (SU1510 model; Hitachi Ltd., Tokyo, Japan) was used to observe the morphology of prepared CS NPs. Particle size and size distributions [polydispersity index (PDI)] were measured by Zetasizer (Malvern Instruments, UK).

Assessment of the drug efficacy

Parasitological examination

• For estimation of parasite count, at the 11th and 19th days post infection (PI) fecal samples were gathered from infected mice. The samples were weighed, then dissolved in 1 ml of 7% formalin and passed through sterile gauze. To count the number of oocysts of Cryptosporidium, 50 μl were taken from each sample, then stained with the modified Ziehl–Neelsen stain (Garcia 2007). The number of parasites was expressed per gram of feces (Benamrouz et al. 2012).

  The % reductions in the count of the parasite were calculated according to this equation: %R = 100(C − E)/C.

  %R: % reductions, C: control group and E: experimental groups of mice (Penido et al. 1994).

• for detection of the mortality rate (MR) (Eissa et al. 2012):

  $$\text{MR}=\frac{\text{Number of dead mice at the sacrifice time}}{\text{Number of mice at the beginning of the experiment}}\times 100$$

Histopathological examination

Specimens from intestine, liver and lung were collected and fixed in 10% formaldehyde. In arising grades of ethyl alcohol, the tissues were dehydrated, then cleared in xylene and immersed in paraffin. Then microtomy was performed to obtain sections of 5µ thick, which were deparaffinized in xylene and rehydrated in descending graded alcohol then stained by haematoxylin and eosin (H&E) stain (Drury and Wallington 1980).

Statistical analysis

Data were presented as mean ± SD using SPSS version 25. ANOVA F-test was used to compute difference between several quantitative variables among subgroups while Student’s t-test was used for comparison between two quantitative variables in the same group. For qualitative data, Fisher’s exact test was used (Kirkwood 2003). p value equal or less than 0.05 indicates significant results (Leslie et al. 1991).

Results

Characterization results

CS NPs have approximately spherical form, smooth surface and their range of size was about 20–40 nm. By SEM, NTZ loaded CS NPs were smooth round in shape with average size about 60–70 nm Fig. 1a, b.

Fig. 1.

Characterization of CS NPs, the size of CS NPs by Zeta sizer (a), and SEM micrograph of NTZ loaded CS NPs (b)

Effect of immunosuppression on murine morphology

In the current work, mice began to express signs of immunosuppression two weeks later from administration of drug in the form of hair loss, subcutaneous edema, skin lesions and ulceration.

Parasitological study

Cryptosporidium oocysts shedding started from the 3rd day post infection (PI) in both immunocompetent and immunosuppressed groups and reached its peak on the 7th day PI. Using modified Ziehl–Neelsen stain, the oocysts appeared as pink bright-rose oval or rounded bodies with variable grades of intensity against a bluish background Fig. 2.

Fig. 2.

Fecal smear stained with modified Ziehl–Neelsen stain showing Cryptosporidium oocysts (black arrows) (×1000)

Treatment with NTZ loaded CS NPs resulted in the highest reduction in the mean oocysts counts per gram feces, followed by treatment with NTZ then CS NPs alone in both days of assessment (11th and 19th days PI).

Table 1 shows that there were high significant statistical differences between all immunocompetent groups in both days of follow up except G2a versus G5a (P3) in both days and G3a versus G4a (P4) at 11th day of follow up. Also there was high significant statistical difference between oocysts counts in both days in groups (G3a) and (G4a) (0.007–0.001) respectively. Additionally, the percentage reduction in oocysts counts was increased with increased day of follow up and the highest percentage reduction of oocysts counts was detected in infected treated NTZ loaded CS NPs mice group (G4a) (75.7%) at 19th day compared to other groups.

Table 1.

Comparison between the oocysts counts in one gram feces in the immunocompetent studied groups

Groups/days	G2a Infected untreated	G3a Infected treated with NTZ	G4a Infected treated with NTZ loaded CS NPs	G5a Infected treated with CS NPs	F^	p
Day 11th Mean ± SD × 103 (R%)	1200 ± 200.13	695.87 ± 77.39 (42.01%)	560.72 ± 9.38 (53.27%)	1090.92 ± 203.68 (9.09%)	42.99	< 0.001**
P within groups		P1 0.001**	P2 0.001** P4 0.26	P3 0.61 P5 0.001** P6 0.001**
Day 19th Mean ± SD × 103 (R%)	1131.81 ± 193.15	485.49 ± 70.28 (57.1%)	275 ± 7.83 (75.7%)	970 ± 174.37 (14.3%)	88.94	< 0.001**
P within groups		P1 0.001**	P2 0.001** P4 0.003**	P3 0.12 P5 0.001** P6 0.001**
Paired t	0.35	3.12	8.24	0.68
p	0.73	0.007**	< 0.001**	0.43

F^: One way ANOVA test, Paired t test

P1: G2a versus G 3a, P2: G2a versus G4a, P3: G2a versus G5a, P4: G3a versus G 4a, P5: G3a versus G5a, P6: G4a versus G5a

*Significant (p < 0.05), **highly significant (p < 0.01)

Table 2 shows that there were high statistical significant differences between all immunosuppressed groups in both days of follow up except G2b versus G5b (P3) in both days and G3b versus G4b (P4) at 11th day of follow up. Also there were high significant statistical differences between oocysts counts in both days in groups (G3b) and (G4b) (0.004–0.001) respectively. Also there was an increase in the percentage reduction of oocysts counts among different groups with increased day of follow up and the highest percentage reduction of oocysts counts was detected in combined therapy group (G4b) (67.33%) at 19th day.

Table 2.

Comparison between the oocysts counts in one gram feces in the immunosuppressed studied groups

Groups/days	G2b Infected untreated	G3b Infected treated with NTZ	G4b Infected treated with NTZ loaded CS NPs	G5b Infected treated with CS NPs	F^	p
Day 11th Mean ± SD × 103 (R %)	1400 ± 456.23	946.15 ± 106.73 (32.42%)	820.57 ± 78.34 (41.39%)	1342.85 ± 172.31 (4.08%)	12.93	< 0.001**
P within groups	–	P1 0.001**	P2 0.001** P4 0.61	P3 0.87 P50.008** P6 0.001**
Day 19th Mean ± SD × 103 (R %)	1272.72 ± 413.87	750.18 ± 96.82 (41.06%)	415.81 ± 70.28 (67.33%)	1175 ± 161.92 (7.68%)	29.67	< 0.001**
P within groups	–	P1 0.001**	P2 0.001** P4 0.01*	P3 0.43 P5 0.002** P6 0.001**
Paired t	0.12	3.15	4.53	0.38
p	0.82	0.004**	< 0.001**	0.65

F^: One way ANOVA test

P1: G2b versus G3b, P2: G2b versus G4b, P3: G2b versus G5b, P4: G3b versus G4b, P5: G3b versus G5b, P6: G4b versus G5b

*Significant (p < 0.05), **highly significant (p < 0.01)

Table 3 there were statistically significant differences between total mortality rate in immunocompetent and immunosuppressed groups. However, the total mortality rate in immunocompetent groups was 8% which show insignificant statistical difference between these groups. Also, the total mortality rate in immunosuppressed groups was 28% with insignificant statistical difference between these groups.

Table 3.

The mortality rate in the different studied groups

Groups	G1 Control (healthy) (N = 20)	G2 Infected untreated (N = 20)	G3 Infected treated with NTZ (N = 20)	G4 Infected treated with NTZ loaded CS NPs (N = 20)	G5 Infected treated with CS NPs (N = 20)	p
a (immunocompetent) N = 50
Lived N (%)	10(100%)	8 (80%)	10(100%)	10(100%)	8 (80%)	0.16
Dead N (%)	0 (0%)	2 (20%)	0 (0%)	0 (0%)	2 (20%)
P within groups	–	P1 0.47	P2 1	P3 1	P4 0.47
b (immunosuppressed) N = 50
Lived N (%)	10 (100%)	6 (60%)	6 (60%)	8 (80%)	6 (60%)	0.17
Dead N (%)	0 (0%)	4 (40%)	4 (40%)	2 (20%)	4 (40%)
P within groups	–	P1 0.09	P2 0.09	P3 0.47	P4 0.09

Fisher’s exact test

P1: G1 versus G2, P2: G1 versus G3, P3: G1 versus G4, P4: G1 versus G5

*Significant (p < 0.05)

Histopathological assessment

Intestine

Sections of intestinal tissues in control infected non-treated immunocompetent and immunosuppressed mice (group 2) showed marked pathological changes in the mucosa due to infection with Cryptosporidium parvum.There were villous atrophy and broadening. Moreover, there were ulceration of intestinal mucosa and inflammatory infiltration of lamina propria with loss of the brush border. In addition, there were proliferation and dysplasia in some mice as shown in Fig. 3b, f. Cryptosporidium parasites were detected at the infected intestinal epithelium Fig. 4.

Fig. 3.

a–e H&E stained intestinal cut sections of immunocompetent mice of different groups. a Section of intestinal tissue in non-infected control group showing normal villous architecture (black arrow), (G1) (×100). b showing (I) atrophy (red arrows), (II) broad villi (black arrows), (III) inflammatory infiltration (green arrow), (G2a) (×100). c Moderate infiltration with inflammatory cells, (G3a) (×400). d In NTZ loaded CS NPs treated immunocompetent group showing normal villous architecture, preserved brush border (red arrow) and mild inflammatory infiltrate (black arrow), (G4a) (×400). e Atrophy and sloughing of mucosa (black arrow) with infiltration of inflammatory cells (red arrow), (G5a) (×100(. f–i H&E stained intestinal cut sections of immunosuppressed mice of different groups. f Abnormal villous architecture (blunted villi) (black arrow) with marked inflammatory infiltration (red arrow), (G2b) (×100). g Glandular proliferation and polyp formation (black arrow), (G3b) (×100). h Mild inflammatory infiltrate (black arrow) and superficial ulceration (red arrows), (G4b) (×100). i Presence of Cryptosporidium parasite at the brush border of epithelial cells of the villi (black arrow) with moderate infiltration of inflammatory cells (yellow arrow) and superficial ulceration (red arrow), (G5b) (×400) (color figure online)

Fig. 4.

Cryptosporidium parasites in the epithelial lining of intestinal villi (black arrows) (H&E × 1000)

Infected mice received NTZ showed moderate histopathological changes while mice received CS NPs alone showed marked changes. Regarding treatment with NTZ loaded CS NPs, there were marked reduction in these changes in the form of improved intestinal mucosa, returning of normal villous pattern with preserved brush border and mild inflammation as shown in Fig. 3c, d, e, g, h, i.

Liver

Liver sections from immunocompetent groups revealed normal liver architecture. While liver sections from infected immunosuppressed control group (G2b) showed disturbed liver architecture by inflammatory infiltration, vacuolar degeneration and hepatocyte atrophy. Mice received CS NPs showed similar changes to infected control group in the form of marked inflammation in liver sections. Mice treated with NTZ (G3b) showed moderate pathological changes in the liver, while mice received NTZ-loaded CS NPs (G4b) showed marked improvement in the liver histopathological pictures in the form of mild inflammatory infiltration and congestion as shown in Fig. 5b, c, d, e, f.

Fig. 5.

H&E stained liver sections of mice of different groups: a Section of liver tissue in non-infected control showing normal liver architecture; central vein (black arrow), normal liver cords (yellow arrow) and sinusoids (green arrow) (× 400). b–f Stained liver sections of immunosuppressed mice of different groups: b disturbed liver architecture by fat vacuoles (black arrows), (G1b) (×400). c showing marked diffuse inflammatory infiltrate (black arrow), (G2b) (×400). d Liver with moderate multifocal inflammatory infiltrate (black arrows), (G3b) (×400). e Improvement of liver architecture but with some congestion (black arrows), (G4b) (×400). f Moderate inflammatory infiltrate (black arrow), binucleated cells (large dysplastic cells) (yellow arrow) and vacuolar degeneration (green arrows), (G5b) (×400) (color figure online)

Lung

Histopathological examination of lung sections from immunosuppressed infected group reveals some moderate pathological changes such as inflammatory cellular infiltration and thick interstitial space. These changes improved with treatment as shown in Fig. 6b, c, d, e.

Fig. 6.

a H&E stained lung section non-infected control group showing normal alveoli (black arrows) (×400). b–e H&E stained lung section in infected immunosuppressed groups. b showing marked inflammation (black arrow) and thick interstitial space (red arrow) (G2b) (×400). c showing moderate inflammatory infiltration of interstitial spaces (black arrow) (G3b) (×400). d showing mild inflammatory infiltration of interstitial spaces (black arrow) (G4b) (×400). e showing moderate inflammatory infiltration of interstitial spaces (black arrow) (G5b) (×400) (color figure online)

Discussion

Cryptosporidiosis is a widespread infection in vertebrates including humans, producing gastroenteritis (Gerace et al. 2019). It is self-limiting in immunocompetent and may be severe and debilitating in compromised patients (Fayer 2008).

CS is a natural polymer widely used in nanomedicine due to its distinctive properties for drug carriage and its NPs are found to be efficient at enhancing drug uptake. The solubility of CS in aqueous medium and its cationic feature have been described as important factors of its success (Akakuru et al. 2018). Also, it can enhance permeation of large molecules through mucosal surfaces (Luppi et al. 2010).

This study was conducted to evaluate and compare the efficacy of NTZ alone and when loaded on CS NPs in the treatment of Cryptosporidium infection.

In this study, dexamethasone was used to induce immunosuppression in the mice as it was considered as a good immunosuppressive agent in numerous studies particularly in mice (Miller and Schaefer 2007). It has high glucocorticoid activity, which has an inhibition effect on the immune response and interferon-gamma pathway (Stojadinovic et al. 2007).

In the present study, number of mice from the immunosuppressed groups showed the development of cutaneous lesions in the form of edema, hair loss and skin ulcers after 14 days of experiment, this may be due to dexamethasone. Various adverse dermatologic effects were associated with glucocorticoid administration including connective tissue and many other dermatological conditions (Siamak Moghadam-Kia et al. 2010). Also Uner et al. (2003) observed many changes in rats after two weeks of immunosuppression, which include petechial haemorrhage of the skin on the ears associated with exophthalmia.

In the current study, shedding of the oocysts started from the 3rd day PI in both immunocompetent and immunosuppressed groups. This agrees to some extent with Chai et al. (1999) who stated that the beginning of oocyst excretion was on 4th day PI in both immunocompetent and immunosuppressed rats. El Shafei et al. (2018) also found that Cryptosporidium oocysts appeared in feces of immunocompetent mice on the 4th day PI while in immunosuppressed mice appeared on the 2nd day PI.

The Cryptosporidium oocysts continued to shed throughout the experiment in infected groups. This result is in agreement with Abdou et al. (2013) who mentioned that shedding of oocysts continued until day 30 PI in infected mice with C. parvum. In addition, Lacroix et al. (2001) informed that the duration of oocysts excretion was extended to the 4th week PI.

In this work, it was found that the shedding of oocysts was higher in immunosuppressed infected mice compared to immunocompetent groups. Similar findings have been informed by Abdou et al. (2013) and Aly et al. (2017).

Regarding the parasitological effect of NTZ, NTZ loaded CS NPs and CS NPs in immunocompetent groups, it was found that treatment of Cryptosporidium infected mice with NTZ loaded CS NPs resulted in the highest percentages of reduction in oocyst shedding (53.27%, 75.7%) when detected on days 11th and 19th PI respectively in comparison to their corresponding infected control group (Table 1).

In immunosuppressed groups, it was also found that treatment with NTZ loaded CS NPs resulted in the highest percentages of reduction in oocyst shedding (41.39%, 67.33%) on days 11th and 19th PI respectively (Table 2).

Sedighi et al. (2016) stated that treatment of C. parvum infection in neonatal rats with NTZ loaded on solid lipid nanoparticles (SLN) was more effective than free drug in reducing the parasite number. Kayser (2001) reported that combination of bupravaquone with CS nanosuspension augmented bupravaquone effect against cryptosporidiosis as it can reside in the intestine for long time.

These findings were in accordance with Mohamed et al. (2019) who stated that using Nigella sativa conjugated with CS NPs was highly effective in reduction of Cryptosporidium oocysts excreted in both immunocompetent and immunosuppressed mice at 27th day PI with percentage reduction (79.16%,73.33%) respectively.

This may be due to the mucoadhesive character of CS and CS NPs. This character increased resident times with a prolonged action and reduced elimination in the gut. CS NPs tend to stick to the intestinal wall, thus they can directly interact with the pathogen in infected gastrointestinal tract (Said et al. 2012).

These findings were also in agreement with Sadek et al. (2018) who found that loading praziquantel (PZQ) on CS NPs improves PZQ effectiveness on Schistosoma premature stages and increased its effect on adult stages even in its half ordinary dose. Moreover, treatment of murine Schistosoma mansoni infection by N. sativa loaded CS NPs reduced the worm burden by 77.5% (Elawamy et al. 2019). Also, it was found that spiramycin loaded CS NPs significantly reduced the number of Toxoplasma parasites in tissues in a study conducted by Etewa et al. (2018).

Regarding the effect of NTZ on cryptosporidiosis in the current study, it was found that there was significant reduction in oocyst shedding in both immunocompetent and immunosuppressed mice. The mean percentages reduction in NTZ treated groups on 11th and 19th days PI were (42.01%, 57.1%) respectively in immunocompetent group and (32.42%, 41.06%) in immunosuppressed group.

These results agreed with that of Amadi et al. (2002) who stated that NTZ resulted in resolution of diarrhea among the patients not infected by HIV (56%) while there were no significant differences in clinical and parasitological responses with NTZ treatment in the HIV infected patients. Rossignol et al. (2001) recorded higher percentage reduction (80%) in non-immunodeficient adults and children with cryptosporidial diarrhea in the Nile Delta of Egypt four days after full course of NTZ treatment.

Gargala et al. (2013) recorded reduction in oocyst shedding by (47%) in NTZ treated immunosuppressed mongolian gerbils. Moreover, Madbouly et al. (2017) stated that NTZ resulting in percentage reduction of (26.8%, and 52.5%) on 11 and 19 days PI respectively in Cryptosporidium infected immunosuppressed mice.

On the contrary, Theodos et al. (1998) reported that no significant difference in oocysts excretion was observed between groups of mice treated with NTZ and control group. Mostafa et al. (2018) reported lower reduction percentages in oocyst shedding among the immunosuppressed groups (10.8% and 8.2%) on days 14 and 21 respectively, that was explained by different drug formulations, doses and different animal models used.

In the present study, regarding the effect of CS NPs on Cryptosporidium infection in immunocompetent mice, it was found that CS NPs resulted in percentage reduction in oocysts count in feces of (9.09%, 14.3%) on 11th and 19th PI respectively in comparison with their corresponding infected control mice (Table 1). While in immunosuppressed groups, it resulted in non-significant reduction in the mean oocysts count/ gm feces (4.08%, 7.68%) at 11th and 19th days PI respectively (Table 2).

The current results were nearly similar to Mohamed et al. (2019) who reported that CS NPs resulted in reduction of oocysts shedding at 18 day PI by 17.3% in immunocompetent group and 11.7% in immunosuppressed mice. Also Aydogdu et al. (2019) reported that CS significantly decreased Cryptosporidium oocysts count compared to the infected untreated group. But, it was not efficient to eliminate the infection completely. Etewa et al. (2018) recorded that treatment of mice infected with Toxoplasma RH strain using CS NPs resulted in reduction of the parasite count by 6.42%, 23.94% and 17.66% in the brain, liver and spleen respectively.

On the other hand, Mammeri et al. (2018) reported that CS NAG (N-acetyl-D-glucosamine) and CS mix when used to test their anticryptosporidial properties in CD-1 mice, it produced 34.5% and 56% inhibition of infection when compared to infected control group. This may be explained by different forms of CS used, different methods of CS preparation, dose and different models used. In addition, infection kinetics are influenced by many factors: the strain of mice, animal age, the Cryptosporidium species used and its strain, infectivity, the infectious dose and the techniques used to detect and count the parasite (Finch et al. 1993).

In this study, there were no detected deaths in immunocompetent groups treated with NTZ alone (G3a) and NTZ loaded CS NPs (G4a), whereas in control infected (G2a) and CS treated (G5a) immunocompetent groups; the mortality rate was 20% in both groups. In immunosuppressed mice, it was found that group treated with NTZ loaded CS NPs showed lower mortality in mice (20%) compared to other groups (Table 3). This agreed with Etewa et al. (2018) who reported that spiramycin-loaded chitosan nanoparticles (SLCNs) reduced the mortality rate of mice infected by toxoplasma significantly in comparison to 100% mortality of mice in the infected control groups.

There was statistically significant difference between total mortality rate in immunocompetent and immunosuppressed groups with (p value = 0.02*) as the mortality rate in immunosuppressed groups was higher than immunocompetent groups (28%, 8%) respectively. These findings coincided with those of Mead et al. (1991) who reported that deaths in immunosuppressed group apparently resulted from hepatic dysfunction. Also, the present results were in agreement with Sadek and El-Aswad (2014) and El Shafei et al. (2018) who recorded higher deaths in immunosuppressed mice.

The histopathological changes in the intestine, the liver and the lung were assessed to evaluate the therapeutic effects of different drug forms on intestinal and extra-intestinal cryptosporidiosis.

In this study, histopathological examination of the intestinal tissue of the control infected groups revealed marked pathological changes in the intestinal mucosa in comparison with the non-infected control groups. There were villous shortening and atrophy, mucosal ulceration, marked inflammation and loss of brush border. Intestinal dysplasia was also detected. These findings were in accordance with Waters and Harp (1996) and AbuEl Ezz et al. (2011). Also, Abdou et al. (2013) and Abdelhamed et al. (2019) reported that Cryptosporidium infection may induce dysplastic changes of intestinal tract in mice.

Similar histopathological findings were reported by Soufy et al. (2017) who reported that Cryptosporidium parasite displacing brush borders resulting in shortening and broadening of the villi. Villous atrophy may be explained by toxins secreted by the pathogen that directly damage the epithelial cells.

Histopathological examination of intestinal sections from mice received NTZ alone showed partial improvement in the pathological changes following Cryptosporidium infection. This was in accordance with that of Sadek and El-Aswad (2014) who detected moderate changes in NTZ treated mice and severe pathological changes in infected control group.

In the current work, mice treatment with NTZ loaded CS NPs showed high improvement in the intestinal histopathological changes. 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.

These findings were in agree with Abdelhamed et al. (2019)who detected improvement of the intestinal pathological changes in the form of mild infiltration of inflammatory cells and detected few Cryptosporidium parasites at the intestinal epithelium of mice treated with nanazoxide combined with artesunate loaded polymeric nano-fiber.

Regarding histopathological changes in the liver, it was found that liver tissues from immunocompetent groups were normal while in immunosuppressed, liver sections from infected control group revealed some pathological changes such as inflammatory cellular infiltration, vacuolization and atrophy of liver cells. Treatment with NTZ loaded CS NPs (G4b) resulted in improvement of the liver picture in the form of mild inflammation and congestion while in NTZ-treated group (G3b) the pathological changes were moderate.

These results were in accordance with Stephens et al. (1999) who reported that chronic Cryptosporidium infection of the biliary tract in severe combined immunodeficiency mice developed cholangitis and lobular hepatitis. Furthermore, hepatic dysplasia following C. parvum infection has been described in severe combined immunodeficiency mice Certad et al. (2012). Abdou et al. (2013) also detected large cell dysplasia in the liver of immunosuppressed group.

Mahmood et al. (2016) also reported that liver section from Cryptosporidium infected immunosuppressed mice showed inflammatory changes with no fibrosis and these changes were improved with miltefosine therapy.

Respiratory cryptosporidiosis has been described in immunodeficient people; most of them were co-infected with HIV (Certad et al. 2010). In the current work, lung tissue examination of immunosuppressed infected mice revealed marked inflammation and thick interstitial spaces. The mice treated with NTZ loaded CS NPs showed improvement in the form of mild inflammation.

Similar findings were detected by Sponseller et al. (2014) and also by Madbouly et al. (2017) who reported that there are pulmonary hemorrhage and interstitial inflammations in the lung tissue of the infected immunosuppressed mice and these changes improved following combination treatment with NTZ and atorvastatin.

Conclusion

Loading NTZ on CS NPs improved NTZ efficacy on cryptosporidiosis in both immunocompetent and immunosuppressed mice. Also, NTZ loaded CS NPs showed better results than free NTZ in reduction of Cryptosporidium oocysts shedding and improving histopathological changes caused by Cryptosporidium infection in the liver, intestine and lung of mice.

Author contributions

All authors contributed to the study conception and design. Material preparation was perfomed by Howayda Said Fouad Moawad, data collection and analysis were performed by Maha Saber Reda Badawey, Amira Abd El-Lateef Saleh Ali and Shereen Mahmoud Ibrahim. The study was finalized by Mohamed Hegab Abd El-Hady Hegab. The first draft of the manuscript was written by Shaimaa Elsayed Ashoush and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Compliance with ethical standards

Conflict of interest

Authors state that they have no conflict of interest.

Ethical aspects

The study was accepted by the Research Ethics Committee, Faculty of Medicine, and Zagazig University. All techniques related to animal experimentation in this study met the International Guiding Principles for Biomedical Research Involving Animals as issued by the International Organizations of Medical Sciences.