*Corresponding author: Sadiya Aziz Anah Department of Biology, Faculty of Education, University of AL-Qadisiyah, Al Diwaniyah 58002, Iraq Introduction
Cryptosporidium parvum is one of the causes of watery diarrhea, whether in humans or animals. This disease causes cryptosporidiosis, which is widespread in varying proportions in developing and developed countries, while some sources indicate high rates of infection in immunosuppressed people [1,2]. C. parvum is also considered a parasite that can reduce growth and lead to mental weakness in infected children at an early age [3,4].
Nanotechnology is one of the most important technologies in modern science and has progressed rapidly. The comprehensiveness and breadth that characterize this technology have met many needs and life requirements [5]. Nanoparticles have unique properties in electronic, magnetic, and chemical catalysis because of their high stability, low reactivity, relatively low toxicity, and multiple forms. These properties have broad applications in various fields of biomedicine, such as the diagnosis and treatment of cancers and other diseases, and industry drugs and gene delivery, and some nanoparticles also have antimicrobial properties for viruses, bacteria, and fungi [6]. Some nanoparticles have been used as antidotes for many pathogens, including protozoan parasites [7,8]. For example, a previous study [9] reported the ability of silver nanoparticles to penetrate the wall of the oocysts of Cryptosporidium, indicating a decrease in the viability of the oocysts at concentrations higher than 0.5 mg/mL.
Zinc is a mineral essential to human health. It is non-toxic but effective even at low concentrations. This mineral plays an important role in most of the body’s biological activities, such as phagocytosis and the production of antibodies and cytokines [10-12]. Considering previous research that indicated the effectiveness of zinc oxide nanoparticles (ZnO-NPs) against Eimeria tenella [13]. In addition, Dkhil et al. [14] reported that giving ZnO-NPs to mice infected with Eimeria papillata helped reduce the harmful effects of oxidative stress. On the other hand, Nazir et al. [15] revealed the effectiveness of zinc nanoparticles against Leishmania tropica, because of the release of reactive oxygen species., Vaidya et al. [16] also examined the effects of silver and ZnO-NPs on the oocysts of C. parvum under different conditions. They reported, that zinc oxide particles were effective in dissolving oocysts and were comparable to freezing and thawing methods. The current study examined the ability of this nanomaterial to oocysts of the C. parvum in experimentally infected mice.
Materials and Methods
Ethical approval
This study was approved by the Committee of the Department of Biology, Faculty of Education, University of AL-Qadisiya (approval number: 578).
Isolation and purification of oocysts of C. parvum
Oocysts of the C. parvum were obtained from some patients who visited Al Diwaniyah Teaching Hospital during the period from 1/11/2024 to 1/1/2025. One hundred stool samples were collected and diagnosed microscopically by making direct smears and stained with modified Ziehl-Neelsen stain.
Oocysts were isolated from stool samples preserved with a potassium dichromate solution using a floatation method and a diabetic solution according to the method reported elsewhere [17]. The number of oocysts per 1 mL of suspension was also calculated using a hemocytometer slide, where the number of oocysts was counted in the eight squares according to the equation below:
Molecular study
Oocysts were diagnosed microscopically using the modified acid stain method. The diagnosis was confirmed molecularly using nested-polymerase chain reaction (N-PCR) technology, where the DNA was extracted according to the method attached to the Taiwanese Presto stool DNA Extraction Kit (Geneaid, Taiwan). The concentration and purity of the extracted DNA were measured using a Nanodrop spectrophotometer by reading the absorbance at 260 to 280 nm. The extracted DNA was stored at 20°C in a refrigerator until a PCR examination was performed. The PCR mixture was then prepared, and the extracted DNA was amplified using special primers to amplify the heat shock protein (HSP70) gene, which was responsible for diagnosing the C. parvum, using N-PCR technology, where two pairs of primers were prepared from Bioneer. The first round of PCR was performed using a pair of C. parvum-HSP70 gene-specific primers: HSP70-F1 5ʼ-TGAGGGTG AGAGAGCCATGA-3ʼ) and HSP70-R1: (5ʼ-GCATACCAC CCT CAG CAGA G-3ʼ). Nested-PCR was performed with a second pair of primers: HSP70-F2 (5ʼATTC CACCAGCAC CA AGA GG -3ʼ, HSP70-R2(5ʼCAGTTTGGTTGTGCTCGAGC-3ʼ). Subsequently, the PCR mixture was transferred to a PCR Thermocycler to perform thermal cycling, after which the PCR result was read by electrophoresis using a 1.5% agarose gel. The gel containing the PCR product was examined using an ultraviolet source to determine the presence of the product with the Ladder unit.
Preparation of ZnO-NPs and infection of the animals
The nanocomposite (ZnO-NPs) was prepared in the form of a stock solution. One gram of ZnO-NPs was dissolved in a liter of distilled water and sterilized in an autoclave. The solution was then mixed using an ultrasonic homogenizer for 15 minutes to prevent the agglomeration of these materials, and the concentrations were prepared at 5, 15, and 20 mg/kg. Thirty Swiss Balb/C mice, aged 21 days and weighing 20 to 25 g, were used. The mice were placed in plastic cages prepared for this purpose in the animal house of the Department of Biology at Al-Qadisiya University, and the appropriate conditions were provided: temperature, ventilation, lighting, water, and continuous feeding. Before starting to dose the mice, their absence of intestinal parasites was confirmed by examining the stool. The, mice were divided into six groups containing five mice each. Groups 1, 2, and 3 in which mice were dosed with 1 mL of the specified dose 1 × 103 oocysts/mouse from suspended oocysts of the parasite C. parvum and then dosed orally with 5, 15, 20 mg/kg of ZNOPs, respectively. The mice in group 4 were dosed with 1 mL of the specified dose: 1 × 103 oocytes/mouse from suspended oocysts of C. parvum and then dosed with 10 mg/kg of nitazoxanide. Group 5 represented the negative control group, dosed with 1 mL of physiological saline, and group 6 comprised the positive control group, dosed with 1 mL of the specified dose 1 × 103 oocysts/mouse from suspended oocysts of C. parvum parasite and left untreated. The feces of mice were examined starting from the second day after dosing until the oocysts appeared to confirm the presence of oocysts. The oocysts were counted after isolating and purifying them, and the therapeutic efficacy was calculated according to Xiao et al. [18].
Results
The results of the experimental infection for laboratory mice administered a dose of 1 × 103 oocysts/laboratory mouse. The rate of infection with the C. parvum reached 100%. The infection of the mice was confirmed by examining the stools using the modified Ziehl- Nelson stain, where the oocysts were observed, which appeared spherical or oval in shape and red in color (Fig. 1). The, oocysts were also confirmed by N-PCR, as shown in Fig. 2. The oocysts were observed on days four and five after dosing (prepatent period).
The rate of excretion of oocysts of C. parvum varied after treatment (Table 1) because the number of excreted oocysts decreased as the concentration and treatment period were increased. Mice infected with the oocysts and treated with zinc nanoparticles at 5 mg/kg continued to excrete oocysts throughout the seven-day treatment period, achieving treatment efficiencies of 4.03%, 9.89%. 15.48%, 25.33%, 28.00%, 59.72%, and 45%, respectively . At 15 mg/kg, a noticeable decrease in the number of excreted oocysts was observed, especially on the sixth and seventh days of treatment, because the number reached 600 oocysts/mL, with 96.31% and 100% efficiency, respectively. At 20 mg/mL the number of excreted oocysts decreased from the first day of treatment, with the mice completely stopping excreting oocysts on the fifth, sixth, and seventh days of treatment, with a therapeutic efficiency of 23.04%, 39.06%, 83.67%, 93.81%, 100%, 100%, and 100%, respectively , with significant differences between the groups. Hence, 20 mg/mL is considered the most efficient concentration. The results from the group treated with nitazoxanide and the control groups were at the probability level of p ≤ 0.05.
Discussion
Cryptosporidiosis is a zoonotic pathogen and a health problem in humans and animals [19]. Nitazoxanide is the only and most widely approved treatment by the Food and Drug Administration [20] because it inhibits the enzyme pyruvate ferredoxin oxidoreductase in the anaerobic metabolic pathway. This drug is ineffective for malnourished children and immunocompromised individuals [21].
Zinc nanoparticles have gained an important place in various medical and pharmaceutical sciences, in addition to their potential use as anti-parasitic agents, owing to their bio-catalytic activities [22]. These antimicrobial properties may be manifested in two ways: by stimulating programmed cell death by inhibiting cell growth, or by zinc nanoparticles producing H2O, which leads to increased oxidative stress, release of zinc ions, and penetration of the cell wall [23,24].
The mice were infected with C. parvum, no other species of the genus Cryptosporidium, because it infects humans and other mammals. The results showed that an experimental infection occurred in all groups dosed with the parasite. Most cases of infection were of the mild type because the number of oocysts ranged between one and four cysts in the prepared swabs. The first oocyst appeared in the feces of mice on days four to five after dosing, which concurs with Khashan et al. [24] It is not consistent with the results of [25], which indicated the appearance of oocysts at a high rate on the second day of infection. HSP70 was chosen because it plays a role in protein folding and transport, as well as the assembly and disassembly of protein complexes, which affects the thermotolerance of animals.
The results of the current study indicated the use of zinc nanoparticles as a treatment for mice infected with oocysts for one week. The effects of the three concentrations (5, 15, and 20 mg/kg) varied in reducing or decreasing the number of oocysts excreted in the feces of the experimentally infected mice, where 20 mg/kg was the best in completely reducing the oocysts on the fifth day of treatment with a therapeutic efficiency of 100%, i.e., the mice completely stopped producing oocysts compared to the positive control group, which continued producing oocysts until the seventh day. This concentration was also closer to the group treated with 10 mg/kg of nitazoxanide, which achieved 100% therapeutic efficacy on the fourth day of treatment. These results are similar to those reported by Vaidya et al. [16], who confirmed that ZnO-NPs were more effective in dissolving the oocysts of C. parvum than silver nanoparticles. These results are similar to those of Hamdy et al. [26], who used ZnO-NPs loaded with nitazoxanide and Allium sativum to treat experimental cryptosporidiosis.
The effectiveness of zinc nanoparticles may be due to their greater solubility, which was confirmed by Kermanizadeh et al. [27]. Zinc nanoparticles are characterized by their ability to penetrate the wall of the oocysts and disrupt the glycoproteins present in the wall of the oocyst, in addition to the small size of these particles that enables them to penetrate the cell membrane and thus cause direct damage [28-30]. Czyżowska amd Barbasz [31] reported that zinc particles bind to the DNA of C. parvum, which affects metabolic processes.
At 15 mg/kg, therapeutic efficiency was achieved on the sixth and seventh days with a therapeutic efficiency of 96.31% and 100%, respectively, while the mice treated with 5 mg/kg continued to excrete oocysts until the seventh day of treatment. This may be because 5 mg/kg is too low compared to 15 mg/kg and 20 mg/kg. In general, an inverse relationship was observed between the rate of the number of oocysts excreted in the feces of infected mice and the concentrations used.
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