Silver nanoparticles as a therapeutic agent in experimental cyclosporiasis

Journal Pre-proof Silver nanoparticles as a therapeutic agent in experimental cyclosporiasis

M.R. Gaafar, L.A. El-Zawawy, M.M. El-Temsahy, Th.I. Shalaby, A.Y. Hassan PII:

S0014-4894(19)30298-X

DOI:

https://doi.org/10.1016/j.exppara.2019.107772

Reference:

YEXPR 107772

To appear in:

Experimental Parasitology

Received Date:

02 July 2019

Accepted Date:

05 October 2019

Please cite this article as: M.R. Gaafar, L.A. El-Zawawy, M.M. El-Temsahy, Th.I. Shalaby, A.Y. Hassan, Silver nanoparticles as a therapeutic agent in experimental cyclosporiasis, Experimental Parasitology (2019), https://doi.org/10.1016/j.exppara.2019.107772

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.

Journal Pre-proof

Title: Silver nanoparticles as a therapeutic agent in experimental cyclosporiasis Gaafar M.R. a*, El-Zawawy L.A. a, El-Temsahy M.M. a, Shalaby Th.I. b, Hassan A.Y. a a

Department of Medical Parasitology, Faculty of Medicine,

b

Department of Medical

Biophysics, Medical Research Institute, Alexandria University, Egypt.

*Corresponding author at: Department of Medical Parasitology, Faculty of Medicine, Egypt. E-mail address: [email protected] (M.R. Gaafar)

Journal Pre-proof

Abstract Cyclosporiasis is an emerging worldwide infection caused by an obligate intracellular protozoan parasite, Cyclospora cayetanensis. In immunocompetent patients, it is mainly manifested by self-limited diarrhea, which is persistent and may be fatal in immunocompromised patients. The standard treatment for cyclosporiasis is a combination of two antibiotics, trimethoprim and sulfamethoxazole. Gastrointestinal, haematologic and renal side effects were reported with this combination. Moreover, sulfa allergy, foetal anomalies and recurrence were recorded with no alternative drug treatment option. In this study, silver nanoparticles were chemically synthesized to be evaluated for the first time for their anticyclospora effects in both immunocompetent and immunosuppressed experimental mice in comparison to the standard treatment. The effect of silver nanoparticles was assessed through studying stool oocyst load, oocyst viability, ultrastructural changes in oocysts, and estimation of serum gamma interferon. Toxic effect of the therapeutic agents was evaluated by measuring liver enzymes, urea and creatinine in mouse sera. Results showed that silver nanoparticles had promising anti-cyclospora potentials. The animals that received these nanoparticles showed a statistically significant decrease in the oocyst burden and number of viable oocysts in stool and a statistically significant increase in serum gamma interferon in comparison to the corresponding group receiving the standard treatment and to the infected non-treated control group. Scanning electron microscopic examination revealed mutilated oocysts with irregularities, poring and perforations. Biochemical results showed no evidence of toxicity of silver nanoparticles, as the sera of the mice showed a statistically non-significant decrease in liver enzymes in immunocompetent subgroups, and a statistically significant decrease in immunosuppressed subgroups. Furthermore, a statistically non-significant decrease in urea

Journal Pre-proof

and creatinine was recorded in all subgroups. Thus, silver nanoparticles proved their effectiveness against Cyclospora infection, and this will draw the attention to its use as an alternative to the standard therapy. Keywords: Cyclospora, experimental mice, treatment, silver nanoparticles.

Journal Pre-proof

1. Introduction Cyclospora cayetanensis (C. cayetanensis) is an emerging intestinal coccidian protozoan that has been considered as an important cause of endemic or epidemic diarrheal illness worldwide (Chacin-Bonilla, 2010). Although man is the only natural host for this infection, the role of animals as natural reservoirs is uncertain but of increasing concern. Human-to-human spread of the parasite occurs indirectly via the environment through ingestion of sporulated oocysts in contaminated food and water (Curry and Smith, 1998; Chacin-Bonilla, 2010). Oocysts were recovered in stools of both immunocompromised and immunocompetent children and adults, with or without a recent travel history (Curry and Smith, 1998; ChacinBonilla, 2010; Chacin-Bonilla, 2012). Several outbreaks associated with C. cayetanensis have been reported in developing as well as developed countries, thus, it is a global public health problem (Chacin-Bonilla, 2012). In Egypt, it was reported for the first time in AIDS patients from Alexandria by Awadalla et al. (1995). Cyclospora infection in immunocompetent patients is manifested by sudden onset of explosive diarrhea, intermittent abdominal cramping, anorexia, weight loss, nausea and vomiting. Diarrhea is self-limited and lasting for one to three weeks. The health risk associated with the disease is usually confined to adult foreigners visiting endemic regions. Consequently, C. cayetanensis causes "traveler's diarrhea" (Chacin-Bonilla, 2010; Bednarska et al., 2015). Besides, the infection in immunocompromised patients leads to prolonged or persistent diarrhea which may be fatal and accompanied by high potentiality of recurrence. Extraintestinal complications as acalculous cholecystitis, thickened gallbladder and elevated

Journal Pre-proof

alkaline phosphatase have been reported. Moreover, the parasites have been identified throughout the gut and the respiratory tract (De Górgolas, 2001; Zar et al., 2001). The specific drug treatment for cyclosporiasis is the combination of two antibiotics, trimethoprim (TMP) and sulfamethoxazole (SMX) (co-trimoxazole). TMP blocks the production of tetrahydrofolic acid, while SMX inhibits the synthesis of dihydrofolic acid of the parasite. Therefore, this combination blocks two consecutive steps in the biosynthesis of nucleic acids and proteins which are essential to the parasite (Gleckman et al., 1981; Kalkut, 1998). Although relief of symptoms has been seen in one to three days post treatment, recurrence occurs within one to three months in over 50% of the patients (Pape et al., 1994; Garcia, 2016). Furthermore, the current use of this combination is associated with gastrointestinal, haematologic and renal side effects especially in immunocompromised patients (Bernstein, 1975; Smith et al., 1980). Moreover, this drug can give rise to further concern due to sulfa allergy in some people (Marinac and Stanford 1993; Wanat et al., 2009). Additionally, the TMP cannot be used during the first trimester of gestation because of the antifolate effect which may have additional detrimental consequences during early foetal development. Meanwhile, SMX can persist in the neonatal circulation for several days after delivery if taken near-term with high potentials for hyperbilirubinaemia and kernicterus in newborns (Lee et al., 2008). Unfortunately, no alternative drug treatment option has been discovered yet for patients who are allergic to sulfa, for pregnant patients, or for those who do not respond to this antibiotic combination (Curry and Smith, 1998). In the present century, a tremendous impetus has been given to the research and applications in the field of nanoscience and nanotechnology to achieve progress in the diagnosis and treatment of different diseases. One of the main focuses of nanotechnology is

Journal Pre-proof

synthesizing

therapeutic

agents

in

biocompatible,

polymeric,

and

minimal-sized

nanocomposites such as nanoparticles (NPs) which possess defined physicochemical and mechanical properties. Among these particles, the metallic NPs became of current interest to researchers due to their proved anti-bacterial properties that are synchronized with development of resistance of several pathogenic bacteria against various antibiotics. Different types of metallic NPs like copper, titanium, zinc, magnesium, gold and silver have come up, among which, silver nanoparticles (Ag NPs) have been the most widely studied and effective agents (Gong et al., 2007; Rai et al., 2009). Ag NPs have promising applications in medicine as they are used in treatment of wounds and burns. Moreover, they have good antimicrobial efficacy against bacteria, viruses and parasites such as Giardia lamblia, Leishmania tropica, Entamoeba histolytica, Toxoplasma gondii and Cryptosporidium parvum (Allahverdiyev et al., 2011; Said et al., 2012; Gaafar et al., 2014; Saad et al., 2015). In addition, as an efficient antimicrobial agent, these nanoparticles have been widely used for the disinfection of water (Abebe et al., 2015). The promising results of Ag NPs in treatment of these parasitic infections highlighted the possibility of their use in treatment of Cyclospora infection especially in immunocompromised hosts. Therefore, the present study aimed at studying the anti-parasitic effectiveness of Ag NPs in the treatment of Cyclospora infection in experimentally immunocompetent and immunosuppressed mice in comparison to the standard treatment; co-trimoxazole. According to our knowledge, the present work is the first to apply Ag NPs as a therapeutic agent against C. cayetanensis.

Journal Pre-proof

2. Materials and methods 2.1.

Preparation of Ag NPs Materials: Silver nitrate (AgNO3, 99.99%) and trisodium citrate dehydrate (C6H5O7Na3.2H2O, 99.99%) were purchased from Sigma Aldrich (Taufkirchen, Germany). Deionized water was used all over the experiment. Methods: Ag NPs were prepared by the chemical reduction method (Fang et al., 2005). Briefly, 50 ml of 1 mM AgNO3 were heated to boiling. Then, 5 ml of 1% trisodium citrate which acts as a reducing and capping agent were added drop by drop. The solution was heated at boiling point under vigorous stirring until the colour changes to pale yellow which indicates the formation of Ag NPs (Figure 1 a & b). Trisodium citrate reduces silver ions and leads to the formation of metallic silver, which is followed by the formation of metallic colloidal silver nanoparticles (Fang et al., 2005; Oliveira et al., 2005; Neena et al., 2012). The solution was cooled to room temperature and left overnight for complete formation of Ag NPs. To eliminate the free citrate, the solution was centrifuged at 20,000 xg for 30 minutes; the precipitate (Ag NPs) was resuspended in deionized water after discarding the supernatant and re-centrifuged. Ag NPs were washed for three times, then, lyophilized using Christ Alpha 1-2 LD Plus system and weighed. Their final concentration was 100 μg /ml (Fang et al., 2005; Oliveira et al., 2005; Neena et al., 2012). The optical spectrum of the Ag NPs was recorded at the wavelength (200 - 800 nm) using UV-Vis spectrophotometer (Evolution 300, Madison, WI). Their size and the morphology were studied using Transmission Electron Microscopy (TEM) (JEOL-100 CX, Tokyo, Japan). The fourier-transform infra-red (FT-IR) spectrum (frequency: 4000 - 400 cm-1) was obtained by the FT-IR spectrophotometer (Alpha-centauri, Shimadzu, Japan, FT-IR-8400S). The hydrodynamic size, polydispersity index (PDI) and zeta

Journal Pre-proof

potential (ζ) were measured using a nano zeta sizer (Malvern, UK), which measures the diameter of the particles using the Dynamic Light Scattering technique (DLS). This involves the detection of the scattered light from particles suspended in an aqueous solution at a fixed angle 173° at 25°C (Fang et al., 2005; Oliveira et al., 2005; Neena et al., 2012). 2.2.

Stool samples collection Fifty samples were collected from immunocompromised, diarrheic patients attending the out-patients clinics of the Main Alexandria University Hospital, after taking their informed consents. Samples were collected in clean, wide-mouthed, screw-capped containers from April to September 2016, with no predilection for patients’ age or sex (Garcia, 2016). All specimens were preserved in 2.5% potassium dichromate (K2Cr2O7) at 4°C until examined by direct wet saline (NaCl) smear, iodine (I) smear, and formol-ether (H2CO-R–O–R′) sedimentation technique to exclude the presence of pathogens other than Cyclospora. Each concentrated specimen was fixed and stained by safranin stain and modified Ziehl-Neelsen stain for detection of Cyclospora and Cryptosporidia oocysts respectively. The size of the detected oocysts was measured by an ordinary light microscope equipped with an ocular micrometer to exclude Cryptosporidia oocysts (Abou El Naga et al., 1998; Garcia, 2016). This study was approved by the Ethics Committee of Alexandria University (0104837).

2.3.

Oocyst Sporulation Samples positive for Cyclospora oocysts were preserved in 2.5% K2Cr2O7 in covered Petri dishes at room temperature (22 - 30°C). Daily microscopic observation of oocysts was done until sporulation took place after 8 - 14 days. Sporulated oocysts were thus ready for mice infection (Smith et al., 1997).

2.4.

Animal Infection

Journal Pre-proof

The sporulated oocysts were washed three times with distilled water to remove the preservative, centrifuged at 1500 xg for 10 minutes, and counted with a haemocytometer to adjust the infection dose (104 oocysts / mouse in 0.1 ml distilled water) and given orally by gastric gavage (Khalifa et al., 2001). 2.5. Experimental animals This work was carried out on 140 laboratory-bred male Swiss Albino mice aged 3 to 5 weeks, and weighing 20 to 25 g. Animals were housed in well-aerated cages under standard living conditions in the colony room of the Parasitology Department, Faculty of Medicine, Alexandria University. They were maintained on a diet composed of wheat, bread and milk on alternative days, and bedding was changed daily. Before infection, mice stools were parasitologically examined by the conventional techniques to exclude the presence of parasitic infections. This animal study was approved by the Ethics Committee of Alexandria University (0104837) (El Fakhry et al., 1998). 2.6. Animal grouping Mice were divided into 80 serving as control (group I) and 60 as experimental (group II). The control group was further subdivided into 20 non-infected non-treated mice (IA), 20 infected non-treated mice (IB), and 40 non-infected treated mice (IC). Each of the 3 subgroups was further subdivided equally into 2 subgroups; immunocompetent (IA1, IB1 and IC1) and immunosuppressed (IA2, IB2 and IC2). Subgroups IC1 (immunocompetent non-infected treated mice) and IC2 (immunosuppressed non-infected treated mice) were further subdivided equally into 2 subgroups, 10 mice each; IC1a and IC2a received co-trimoxazole, IC1b and IC2b received Ag NPs. Mice of the experimental group (group II) were further subdivided

Journal Pre-proof

into 30 infected treated immunocompetent mice (IIA) and 30 infected treated immunosuppressed mice (IIB). These were further subdivided into 2 equal subgroups, 15 mice each; (IIA1 and IIB1) received co-trimoxazole and (IIA2 and IIB2) received Ag NPs. 2.7. Drugs Three drugs were administered; cyclophosphamide (Endoxan) (Asta Medica AG, Halle/Westfalen, Germany), and two treating drugs; co-trimoxazole; TMP – SMX (Sutrim) (Memphis Co. for Pharm. & Chem. Ind., Cairo, Egypt) and Ag NPs. Mice of subgroups IA2, IB2, IC2 and IIB were immunosuppressed by 2 intraperitoneal (i.p.) doses of cyclophosphamide, 70 mg/kg each, with one-week interval. Forty-eight hours after the second dose, mice of subgroups IB2 and IIB were infected with Cyclospora oocysts (Sherwood et al., 1982). Co-trimoxazole was then administered to subgroups IC1a, IC2a, IIA1 and IIB1 in a dose of 5 mg/kg TMP combined with 25 mg/kg SMX once daily for 7 days starting from the 6th day P.I. (Madico et al., 1997). While subgroups IC1b, IC2b, IIA2 and IIB2 received Ag NPs. The minimal effective and safe dose of Ag NPs and the route of administration (i.p. or oral) of both co-trimoxazole and Ag NPs were determined after a pilot study depending on the oocyst count in mice stool and the biochemical study through measurement of liver and kidney function tests. Three doses of Ag NPs were tried; 5, 10 and 20 μg / mouse, either i.p. or orally once daily for 7 days starting from the 6th day P.I. The dose of 5 μg/ mouse showed low efficacy. The dose of 20 μg / mouse was more efficient than 10 μg /mouse, however, the latter was safer. Furthermore, the i.p. route of administration was better than the oral one. Thus, the selected minimal effective dose was 10 μg /mouse which was given i.p. While for the co-trimoxazole, both oral and i.p. routes of administration showed

Journal Pre-proof

no difference in oocyst count in mice stool, hence the i.p. route was selected to be compatible with that of Ag NPs. Stool samples were collected from infected mice starting from the 6th day P.I every other day till the end of the study (14th day P.I). While blood samples were collected by cervical incision of each mouse on the day of sacrifice, centrifuged and serum samples were separated and kept at - 20 o C until used. All infected mice were sacrificed on the 14th day P.I. As regards subgroup (IC), sacrifice of mice was done 2 days after the last dose of the treating drugs, while mice in subgroup IA2 were sacrificed 16 days after the last dose of the immunosuppressive drug. 2.8. Evaluation of Ag NPs in comparison with Co-trimoxazole 2.8.1. Parasitological study a- Stool oocyst load: Stool samples were pooled at each duration from each infected subgroup (IB, IIA & IIB) separately and smears were prepared. Each smear was stained by 10 µl of safranin stain (1g safranin powder dissolved in 99 ml of distilled water, and 1% methylene blue as counterstain) (Garcia, 2016). Six smears were prepared for each subgroup at each duration. Microscopical examination and counting of Cyclospora oocysts were done in 10 fields of each smear by the high power lens by 3 different examiners and the mean count for each subgroup was calculated. The percentage of reduction (% R) of parasite burden in the infected treated subgroups was estimated as compared with the infected non treated control by the following equation: %R=100 (C-W/C), where C represents the total number of parasites recovered from the infected control subgroup, and W represents the total number of parasites recovered from each infected treated subgroup (Penido et al., 1994).

Journal Pre-proof

b- Viability study: On the 14th day P.I, before sacrifice of animals, smears were prepared from the pooled mice stools of subgroups (IB, IIA & IIB), stained by 0.4% trypan blue stain (w/v) and examined microscopically under the high power lens to count the viable and dead organisms in 10 fields. Viable oocysts appear with clear light blue cytoplasm, while dead oocysts appear with dark blue cytoplasm and unrecognized internal structure. The percentage of viability was calculated by the following equation: Viability % = Mean number of viable oocysts / Mean number of total oocysts X 100 (Turchany et al., 1995; Steenbergen et al., 2001). The % R in parasite viability was also estimated as mentioned before. c- Ultrastructural study: The ultrastructure of Cyclospora oocysts in stool of subgroups (IIA, IIB & IB) was observed by the Scanning Electron microscope (SEM) (Joel JSM- 5300LA, Tokyo, Japan). Specimens were first washed with PBS, fixed in 2.5% glutaraldehyde, then washed twice with double distilled water, and dehydrated in ascending concentration of ethyl alcohol (30% - 100%). This was followed by embedding in epoxy resin, placing on aluminum stubs, and coating with 20 nanometer gold particles (Klainer and Betsch, 1970). 2.8.2. Immunological study Serum Interferon gamma (IFN-γ) was recorded using an IFN-γ ELISA detection kit (Chongqing Biospes Co., Ltd, Chongqing, China) at a wavelength of 450 nm. The assay was carried out as suggested by the manufacturer where the detectable level of the kit is 31.2-2000 pg/ml. 2.8.3. Biochemical study for determination of drug toxicity The effect of treating drugs on liver and kidney functions was studied by measurement of liver transaminases; aspartate transaminase (AST) and alanine transaminase (ALT) by double enzymatic reaction method using Dimension Xpand Plus Integrated Chemistry System

Journal Pre-proof

(Siemens, New York, NY), while serum urea and creatinine were measured by Jaffe reaction method using Dimension Xpand Plus Integrated Chemistry System (Siemens, New York, NY) (Chorawala et al., 2013). 2.9. Statistical analysis Data of the present study were analyzed using IBM SPSS software package version 20.0 (IBM Corp., Armonk, NY). The Kolmogorov-Smirnov test was used to verify the normality of distribution of variables. Quantitative data were described using range (minimum and maximum), mean, standard deviation and median. Mann Whitney test was used to compare between two studied groups for abnormally distributed quantitative variables. While, post-hoc test (Tukey) was used for pairwise comparisons. Significance of the obtained results was judged at the 5% level (Kotz et al., 2006; Kirkpatrick and Feeney, 2013).

Journal Pre-proof

3. Results 3.1.

Results of Ag NPs characterization The TEM micrograph of the chemically prepared Ag NPs is presented in Fig. 2. It was observed that most of the Ag NPs were spherical and homogeneously distributed. There was a variation in particle sizes ranging from 7.47 nm to 15.5 nm with an average size of 10 nm. The UV-Visible spectrum analysis of Ag NPs revealed the surface plasmon resonance (SPR) features of Ag nanostructures. The absorption spectrum of Ag NPs showed a sharp SPR feature at 422 nm (Fig. 3a). FT-IR spectroscopic analysis of Ag NPs showed the absorption band at 2068 cm-1, a sharp narrow peak at 1636 cm-1, and a broad O-H peak at 3349 cm-1 (Fig. 3b). The hydrodynamic diameter of Ag NPs was 19.03 nm (Fig. 3c) and the PDI was 0.412. The zeta potential (ζ) of Ag NPs was - 45.2 mV (Fig. 3d).

3.2.

Selection of positive samples with Cyclospora oocysts Out of the 50 stool samples collected from the patients, examined microscopically using safranin stain and measured by an ocular micrometer, 7 samples with Cyclospora oocysts were detected (14 %). The oocysts appeared spherical, 8 to 10 µm in diameter and stained uniformly orange with a central vacuole (Fig. 4).

3.3. Results of the parasitological study 3.3.1. Parasite count A decrease in the oocyst number was noticed in all infected treated subgroups (IIA1, IIA2, IIB1 and IIB2) throughout the study when compared with the corresponding infected nontreated subgroups (IB1 and IB2). However, the recorded decrease was statistically significant only on the 10th, 12th and 14th day of infection. Furthermore, a % R starting from 6.7 % up to

Journal Pre-proof

96.9 % was noted in the immunocompetent subgroups (IIA1 and IIA2) (Table I), and starting from 17.1 % up to 92.4 % in the immunosuppressed subgroups (IIB1 and IIB2) (Table II). The highest % R was obtained in subgroup IIA2 on the 14th day P.I. On the other hand, when comparing the oocyst number in stool of infected subgroups treated by Ag NPs (IIA2 and IIB2) with those treated by co-trimoxazole (IIA1 and IIB1), the former showed a higher degree of reduction in oocyst shedding with a statistically significant difference between subgroups IIA1 and IIA2 on the 14th day P.I., and between IIB1 and IIB2 on the 10th, 12th, and 14th day P.I. (Table I & II). 3.3.2. Oocyst viability A statistically significant decrease was reported in the number of viable oocysts in all infected treated subgroups (IIA1, IIA2, IIB1 and IIB2) when compared with the infected non-treated subgroups (IB1 and IB2) on the 14th day P.I, with a high % R reaching 100% in Ag NPstreated subgroups (IIA2 & IIB2). Regarding the immunocompetent subgroups, the viability % was 92.3, 72.7 and 0.0 in subgroups IB1, IIA1, and IIA2 respectively. While, in the immunosuppressed subgroups, the viability % in subgroups IB2, IIB1 and IIB2 was 95.6, 88.2 and 0.0 respectively (Table III). When comparing the number of viable oocysts in infected subgroups treated by Ag NPs (IIA2 & IIB2) with those treated by co-trimoxazole (IIA1& IIB1), better results were obtained in the Ag NPs-treated subgroups where complete eradication of the viable oocysts was recorded, with a statistically significant difference between both treated subgroups in both immunocompetent and immunosuppressed animals (Table III) (Fig. 5 a& b). 3.3.3. SEM results

Journal Pre-proof

By SEM (Plate I), the ultrastructure of Cyclospora oocysts was more or less similar in both infected non-treated subgroups whether immunocompetent (IB1) or immunosuppressed (IB2). The oocysts were spherical, with an outer smooth regular fibrillar coat and an indentation on the surface at one side (Fig. a). In subgroup IIA1 (immunocompetent cotrimoxazole-treated), the oocysts appeared shrunken with multiple compressions and pores (Fig. b). While in subgroup IIB1 (immunosuppressed co-trimoxazole-treated), some oocysts still preserved the normal features, and others showed irregularities on their surface with pores, papules and dimples (Fig. c). The changes detected in Ag NPs-treated oocysts were more obvious. In subgroup IIA2 (immunocompetent Ag NPs-treated), massive mutilations were observed in oocysts with distortion in their shape. Some oocysts appeared cauliflowerlike with evident deep perforations throughout their diameters (Fig. d), while others revealed deep clefts and furrows (Fig. e). Meanwhile in subgroup IIB2 (immunosuppressed Ag NPstreated), some oocysts appeared shrunken with obvious deep pores and cavitations (Fig. f). The changes observed in the oocysts of treated subgroups, whether by co-trimoxazole or Ag NPs, were more pronounced in the immunocompetent subgroups. 3.4. Immunological results Regarding the immunocompetent non-infected subgroups, a statistically non-significant decrease and a statistically significant increase in the mean value of serum IFN-γ was observed in the co-trimoxazole-treated subgroup (IC1a) and Ag NPs-treated subgroup (IC1b) respectively in comparison with subgroup IA1 (non-infected non-treated subgroup). The difference between the mean values in both treated subgroups was statistically significant. While in the infected subgroups, a statistically significant increase in the mean value of serum IFN-γ was recorded in subgroup IB1 (infected non-treated) as compared to subgroup IA1

Journal Pre-proof

(non-infected non-treated). In the infected treated subgroups, a statistically non-significant increase in the infected co-trimoxazole-treated subgroup (IIA1) was noted in comparison with the infected non-treated subgroup (IB1). While in the infected Ag NPs-treated subgroup (IIA2), the recorded increase was statistically significant in comparison with subgroup IB1 and subgroup IIA1. On the other hand, as regards the immunosuppressed subgroups, the mean value of serum IFN-γ in both non-infected non-treated subgroup (IA2) and non-infected co-trimoxazoletreated subgroup (IC2a) was beyond the detectable level of the kit used. However, it was detectable in the non-infected Ag NPs-treated subgroup (IC2b). A statistically non-significant increase in the mean value of serum IFN-γ was recorded in the infected co-trimoxazoletreated subgroup (IIB1) when compared to subgroup IB2 (infected non-treated subgroup). Meanwhile, there was a statistically significant increase in the mean value of serum IFN-γ in the infected Ag NPs-treated subgroup (IIB2) in comparison with subgroup IB2 and subgroup IIB1 (Table IV). 3.5.

Safety profile As regards the liver transaminases (AST & ALT) in the immunocompetent subgroups, the changes in all values in both treated and non-treated subgroups either infected or noninfected (IIA1, IIA2, IC1a and IC1b) were statistically non-significant in comparison with the corresponding control subgroups (IB1 & IA1). Similarly, the difference in the mean values between both treated subgroups whether infected (IIA1 & IIA2) or non-infected (IC1a & IC1b) was statistically non-significant. However in the immunosuppressed subgroups, the only statistically significant decrease in the mean values of the liver transaminases was noted in the Ag NPs-treated subgroups whether infected (IIB2) or non-infected (IC2b) when

Journal Pre-proof

compared to the corresponding control subgroups (IB2 & IA2) and the co-trimoxazole-treated subgroups (IIB1 &IC2a respectively) (Table V). Concerning the levels of serum urea and creatinine, the recorded decrease in all values of treated subgroups was statistically non-significant in both immunocompetent and immunosuppressed subgroups whether infected or non-infected in comparison with their corresponding non-treated control. Moreover, the difference between the mean values of serum urea and creatinine in both treated subgroups was also statistically non-significant (Table VI).

Journal Pre-proof

4. Discussion An increased incidence of cyclosporiasis has been reported worldwide among travelers from areas of endemicity and indigenous persons in developing countries. Moreover, outbreaks and sporadic cases have been discovered in developed countries (CDC, 2017). Unfortunately, many side effects were reported with co-trimoxazole. Moreover, in immunocompromised patients, long-term secondary prophylaxis is necessary to prevent recurrence (Bayard et al., 1992; Ortega and Sanchez, 2010). Thus, an effective sulfa-free alternative therapeutic agent is needed for the treatment of the disease. Recently, much attention has been paid towards the use of nanoparticles as an alternative to conventional antimicrobial agents. Among different types of nanoparticles, Ag NPs are superior as they have not only chemical activity against microbes, but they also produce the highest electrical and thermal conductivity of all metals (Nooshin, 2016; Norouzi, 2017). To the best of our knowledge, the current study is the first to evaluate Ag NPs as a therapeutic agent against experimental cyclosporiasis in immunocompetent and immunosuppressed mice in comparison with the standard treatment; co-trimoxazole. In the present study, Ag NPs were prepared by the chemical reduction method which was found to be the simplest method to obtain NPs without aggregation and with high yield and low preparation cost (Szczepanowicz et al., 2010). In addition, active silver ions which are directly proportional to the antibacterial effectiveness of Ag NPs are adsorbed effectively on the surface of chemically synthesized NPs (Ognik et al., 2016). The characterization of Ag NPs in the present work was done by different parameters. The findings of TEM were in agreement with those reported by other researchers. (Gaafar et al., 2014; Malaikozhundana et al., 2016; Midha et al., 2016; Vijayakumar et al., 2016; Zhang et

Journal Pre-proof

al., 2016). The sharp SPR feature detected by the UV-Visible spectrum analysis indicates monodispersity of the sample with no evidence of aggregation and presence of spherical or roughly spherical Ag NPs (Malaikozhundana et al., 2016; Midha et al., 2016; Vijayakumar et al., 2016). Regarding FT-IR spectroscopy, the absorption band at 2068 cm-1 can be ascribed to citrate precursor __ (CH2), while the sharp narrow peak at 1636 cm-1 corresponds to carbonyl stretching (C=O) and the broad O-H peak at 3349 cm-1 is due to water molecules apparent in the sample. These results coincide with Awwad et al. (2013) and Midha et al. (2016) who referred the reduction of silver ions into Ag NPs to the presence of carbonyl (−C=O), and hydroxyl (−OH) groups. Moreover, Zhang et al. (2016) stated that the carbonyl group has a strong binding ability with metal, suggesting the formation of a layer covering Ag NPs and acting as a stabilizing agent to prevent agglomeration in the aqueous medium. Concerning particle size analysis, the high negativity of (ζ) indicates the stability of silver colloidal solution, good colloidal nature and the high dispersity of Ag NPs due to negative repulsion, and the high energy barrier needed for the stabilization of the nanosuspension (Sankar et al., 2013; Mukherjee et al., 2014). Hereby, cyclophosphamide was selected for its specific immunosuppressive activity on both cell-mediated and humoral immunities. It was given in 2 doses prior to infection, which was reported to increase its immunosuppressive efficiency (Hands-chumacher, 1991). Martine (1989) supposed that repeated administration of cyclophosphamide induced better immunosuppression and avoided recovery of the immune response. The dose of cotrimoxazole administered in the present study coincides with the universal dose used in treatment of human cyclosporiasis (160 mg TMP and 800mg SMX twice daily for 7 days) (Madico et al., 1997; Verdier et al., 2000). They reported a significant decrease in oocyst

Journal Pre-proof

excretion. Following the pilot study, co-trimoxazole was administrated i.p. The same route was applied by Yasouka et al. (1995) to treat Pneumocystis carinii by co-trimoxazole in mice. While for Ag NPs, the selected dose was 10 µg /mice i.p. Similar dose and i.p. route of administration was also chosen by Gaafar et al. (2014) to treat the RH strain of T. gondii by Ag NPs. The duration of treatment with co-trimoxazole is known to be 7 days (Verdier et al., 2000). Thus, in the present study Ag NPs were also administrated for 7 days to unify the duration of treatment. Concerning the oocyst count in mice stool, the highest % R was observed in Ag NPstreated subgroups which could be attributed to their mechanisms of action as antimicrobial agents. Sabella et al. (2014) predicted that, once the metal NPs are abundantly taken-up in cells, they are rapidly confined in endosomes and finally in lysosomes. The acidic lysosomal pH triggers a lysosome-enhanced Trojan horse effect (LETH) that combines the abundant cellular internalization of the NPs via active processes with the consequent enhanced release of the relatively toxic ions (silver ions) which may then exert ion-specific toxicity e.g., enzyme depletion/inactivation, protein denaturation against some cellular targets as mitochondria, and/or lysosomal damage or dysfunction. Moreover, Asharani et al. (2009) stated that heavy accumulation of silver ions within the mitochondria impairs their function via oxidative stress. Damage of mitochondrial enzymes leads to cessation of adenosine triphosphate (ATP) synthesis which induces antimicrobial effects. Furthermore, phagocytosis of Ag NPs stimulates inflammatory signaling through the generation of reactive oxygen species (ROS), followed by secretion of tumour necrosis factor (TNF-α) by the activated cells which causes damage of cell membrane and apoptosis (Murphy et al., 2016). The production of high amounts of ROS in response to the released silver ions also causes break in the DNA

Journal Pre-proof

of the organism, stops its replication and leads to its death (Cho et al., 2005). In addition, Xu et al. (2013) suggested that Ag NPs are potent enhancers of the immune system especially cell-mediated immunity, which also plays a role in destruction of intracellular organisms. In the current study, the anti-cyclospora effect of Ag NPs was evaluated for the first time, according to our knowledge. Thereby, no data were available about their effect on oocyst burden. However, their effect was previously evaluated against a number of intracellular and extracellular parasites in several studies and proved their antiparasitic efficacy, and they suggested that the reduced percentage in the parasite count increased by increasing the concentration of Ag NPs (Allahverdiyev et al., 2011; Ponarulselvam et al., 2012; Said et al., 2012; Abebe et al., 2014; Gaafar et al., 2014). Concerning the oocyst viability, the highest % R was observed in Ag NPs-treated subgroups (IIA2& IIB2). This could be attributed to the capability of Ag NPs to produce silver ions which induce production of ROS that have the ability to kill infectious agents (Cho et al., 2005; Murphy et al., 2016). Moreover, Ag NPs have the ability to anchor onto the cell surface and penetrate into the cells, by their small size, through direct diffusion via the pores or ion channels and transporter proteins present in the cell membrane which in turn cause membrane damage and resulting in cell lysis (Ansari et al., 2014). Similar results were reported by Gherbawy et al. (2013) and Cameron et al. (2015) who found reduction in the viability of both Fasciola species eggs and Cryptosporidium parvum oocysts respectively under the effect of Ag NPs. In the present work, the SEM study showed that Cyclospora oocysts appeared nearly similar in both infected non-treated subgroups (IB1&IB2). Similar ultrastructural appearance

Journal Pre-proof

was described by Abou El-Naga (1999). Oocysts of co-trimoxazole-treated subgroups showed minimal changes on their surfaces which may be referred to the mechanism of action of cotrimoxazole that blocks the biosynthesis of nucleic acids and proteins which in turn affects the cell wall of the parasite (Kalkut, 1998). Meanwhile, the changes detected in oocysts of Ag NPs-treated subgroups were more pronounced. These results could be attributed to the interaction of Ag NPs with the surface of the parasites and their ability to anchor to the cell wall and consequently penetrate it, thereby causing structural changes in the cell membrane (Sondi and Salopek-Sondi, 2004). The SEM changes observed in the Ag NPs-treated oocysts may explain the complete loss of the oocyst viability in these subgroups. The effect of both treating drugs; Ag NPs and co-trimoxazole on the ultrastructure of oocysts was more obvious in the immunocompetent subgroups. This might be explained by the combined effect of the drugs and the intact immune cells on the oocysts. Ag NPs produced similar SEM changes on the tachyzoites of T. gondii in the study carried out by Gaafar et al. (2014). Similarly, Saad et al. (2015) and Cameron et al. (2016) reported that Ag NPs induced disorganization of C. parvum oocysts and disruption of their walls. Gherbawy et al. (2013) using SEM also noticed distortion and perforations on the eggs’ surface of Fasciola hepatica when treated with triclabendazole and Ag NPs and these abnormalities were less obvious in those treated with triclabendazole alone. They referred these ultrastructural changes to the interaction of Ag NPs with the surface of the organism. In the present work, IFN-γ was measured in mice sera to determine the effect of Ag NPs on the immune system as it has an important role in recruiting macrophages to sites where antigens are present. It also has a direct inhibitory action on the growth of intracellular organisms (Pollok et al., 2001). Apoptosis induction in infected intestinal epithelial cells and

Journal Pre-proof

modulation of mucosal epithelial integrity are other protective roles of IFN-γ (LacroixLamande et al., 2002). It also activates lysosomal enzymes and induces nitric oxide production which are efficient in elimination of intracellular parasites (MacMicking, 2012). In cryptosporidial infection, IFN-γ was reported to activate enterocytes inhibiting parasite invasion and reproduction and to promote mucosal inflammation eliciting microbicidal processes (Lacroix-Lamande et al., 2002; Choudhry et al., 2009). In the current work, concerning the immunocompetent subgroups, the mean value of IFN-γ in the infected non-treated subgroup (IB1) was significantly higher than that recorded in non-infected non-treated subgroup (IA1). This may be attributed to the IFN- γ production by neutrophils in response to infection with the intracellular parasite (Yun et al., 2000). It was also reported that IFN-γ is secreted by natural killer and T cells as a result of pathogen recognition by Toll-like receptors (TLRs) (Sturge et al., 2013). Meanwhile, a significantly higher level of IFN-γ was observed in the Ag NPs-treated subgroups whether infected or noninfected (IIA2& IC1b) in comparison to non-treated subgroups (IB1& IA1) and cotrimoxazole-treated

subgroups

(IIA1&IC1a).

This

could

be

attributed

to

the

immunomodulatory efficacy of Ag NPs which exerts an adjuvant effect that is mainly ascribed to the recruitment and activation of leukocytes especially macrophages, and the increase in cytokine levels, IgG concentration and phagocytes. This adjuvant effect implies the activation of the immune system by Ag NPs (Xu et al., 2013; Abd AL-Rhman et al., 2016). Moreover, Xu et al. (2013) reported an increase in the number of peritoneal leukocytes and in the MHC II molecules level on the surface of Ag NPs-stimulated macrophages which indicated activation of the antigen-presenting cells (APCs) by Ag NPs. They also recorded an increase in the level of IFN-γ and TNF-α in the abdominal lavage fluid of Ag NPs-treated

Journal Pre-proof

mice, which is responsible for the augmented immune response. The synergistic effect of Ag NPs and the secreted IFN- γ in response to infection can justify the recorded highest % R in the oocyst burden in mice stool and the loss of oocyst viability in the infected Ag NPs-treated subgroups. These results are in agreement with those of Gaafar et al. (2014) who reported increase in the level of IFN-γ in mice infected with T. gondii and treated with Ag NPs. As regards the immunosuppressed non infected subgroups, the mean value of IFN-γ in both non-infected non-treated subgroup (IA2) and the non-infected co-trimoxazole-treated subgroup (IC2a) was beyond the detectable level. This could be due to the immunosuppressive state in these subgroups induced by administration of cyclophosphamide. This was approved by Zoheir et al. (2015) who stated that cyclophosphamide administration down-regulates the IFN- γ genes. However, IFN-γ could be detected in the non-infected Ag NPs treated subgroup (IC2b). Thus, it may be suggested that Ag NPs could ameliorate the immunosuppressive effect of cyclophosphamide, and hence the level of IFN-γ returned to the normal level. Additionally, there was a statistically significant increase in the IFN-γ in the infected Ag NPs-treated subgroup (IIB2) in comparison with the infected non-treated subgroup (IB2) and the infected co-trimoxazole-treated subgroup (IIB1). In spite of the immunosuppressive effect of cyclophosphamide on subgroup IIB2, administration of Ag NPs could overcome this effect and induced a certain degree of stimulation to the immune system which led to elevation of IFN-γ. However, the IFN-γ level in this subgroup was much lower than that in the corresponding immunocompetent subgroup (155 and 485 pg/ml respectively). Hence, the reduction in oocyst count and viability in subgroup IIB2 may be attributed mainly to the direct effect of Ag NPs on the parasite. Moreover, the low level of IFN-γ in subgroup IB2 may explain the recorded high number of oocysts in stool of this subgroup till the end of

Journal Pre-proof

the study as the immunosuppressive state allowed the parasite’s growth, multiplication and subsequently increased its number. Liver and kidneys were identified as the main target organs for nano-silver deposition (Loeschner et al., 2011; Prabhu and Poulose, 2012). Hence, in the present work, the safety of Ag NPs was assessed by measuring liver enzymes, urea and creatinine, and no toxic effects were recorded. It was found that the level of liver enzymes (AST& ALT) increased in the immunosuppressed non-treated subgroups whether infected (IB2) or not (IA2) as well as in the immunosuppressed co-trimoxazole treated subgroups (IC2a & IIBI). This could be attributed to the hepatotoxic effect of cyclophosphamide (Subramaniam et al., 2013). In addition, cyclosporiasis in immunosuppressed individuals is usually associated with acalculous cholecystitis inducing elevation of liver enzymes as reported by Zar et al. (2001). In the present study co-trimoxazole was given for a short duration (7 days), thus it cannot be the cause of elevated liver enzymes which is a well-known side effect of the long term cotrimoxazole treatment (Kowdley et al., 1992). On the other hand, a statistically significant decrease in the level of liver enzymes was found in the immunosuppressed Ag NPs-treated subgroups, whether infected or not, in comparison with the co-trimoxazole treated subgroups and the infected non treated subgroup. Reduced activity of AST and alkaline phosphatase (ALP) was also noted by Ahmadi (2012) in blood plasma of chickens receiving nano-silver, which was referred to the oxidative stress and release of free radicals in the body under the effect of Ag NPs. Moreover, Ognik et al. (2016) reported reduction in activity of the liver enzymes AST and ALT after the administration of nano-silver to the chickens which indicated a disturbance in protein catabolism. As regards the infected subgroup, the decrease could be attributed, from our point of view, to the ameliorating effect of Ag NPs on Cyclospora

Journal Pre-proof

infection and the possible pathological changes accompanying the infection in immunosuppression. In the present work, the capability of Ag NPs to alleviate Cyclospora infection was proved in the form of decreased oocyst burden and viability and hence the expected pathological effect of Cyclospora on the intestine and the accessory organs as gall bladder and liver could be diminished. Consequently the level of liver enzymes returned to normal following administration of Ag NPs. As regards urea and creatinine, they were within the normal range in all studied subgroups. Cyclophosphamide was reported to be reasonably well tolerated by the kidney (Steinberg and Steinberg, 1991). Moreover, co-trimoxazole is a widely used antibiotic for the treatment of urinary tract infections. While, the SMX component appears to be nephrotoxic only at high or incorrectly adjusted doses, especially in hypertensive or diabetic patients (Fraser et al., 2012). Our results regarding absence of any toxic effect of Ag NPs on liver and kidney functions were in agreement with the studies carried out by Youssef et al. (2012), Said et al. (2012) and Gaafar et al. (2014). 5. Conclusion In conclusion, the present study provides evidence that Ag NPs can be considered a promising alternative to the standard therapy, co-trimoxazole in treating cyclosporiasis especially in the immunocompromised host in whom the adverse effect of co-trimoxazole cannot be tolerated. This was emphasized by the capability of Ag NPs to stimulate the immune system and/or ameliorate the immunosuppressive state of the host. However, a further study is recommended to follow up the possible excretion of oocysts in stool after the end of the treatment to investigate the possibility of drug relapse which was previously

Journal Pre-proof

reported with the standard treatment. Another goal may be the use of Ag NPs as a preventive measure against Cyclospora in water sources which also needs further investigations.

Funding sources This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Declaration of interest: None.

6. References -

Abebe, L.S., Su, Y.H., Guerrant, R.L., Swami, N.S., Smith, J.A., 2015. Point-of-use removal of Cryptosporidium parvum from water: Independent effects of disinfection by silver nanoparticles and silver ions and by physical filtration in ceramic porous media. Environ. Sci. Technol. 49 (21), 12958-12967.

-

Abou El Naga, I.F., Negm, A.Y., Awadalla, H.N., 1998. Preliminary identification of an intestinal coccidian parasite in man. J. Egypt. Soc. Parasitol. 28(3), 807-814.

Journal Pre-proof

-

Abou El Naga, I.F., 1999. Studies on a newly emerging protozoal pathogen: Cyclospora cayetanensis. J. Egypt. Soc. Parasitol. 29(2), 575-586.

-

Abd AL-Rhman,

R.M., Ibraheem,

S.R., AL-Ogaidi, I., 2016. The effect of silver

nanoparticles on cellular and humoral immunity of mice in vivo and in vitro. J. Biotechnol. 15(2), 21-29. -

Ahmadi, F., 2012. Impact of different levels of silver nanoparticles (Ag NPs) on performance, oxidative enzymes, and blood parameters in broiler chicks. Pak. Vet. J. 32, 1-4.

-

Allahverdiyev, A.M., Abamor, E.S., Bagirova, M., Ustundag, C.B., Kaya, C., Kaya, F., Rafailovich, M., 2011. Antileishmanial effect of silver nanoparticles and their enhanced antiparasitic activity under ultraviolet light. Int. J. Nanomedicine 6, 2705-2714.

-

Ansari, M.A., Khan, H.M., Khan, A.A., Ahmad, M.K., Mahdi, A.A., Pal, R.,Cameotra, S.S., 2014. Interaction of silver nanoparticles with Escherichia coli and their cell envelope biomolecules. J. Basic Microbiol. 54(9), 905-915.

-

Asharani, P.V., Low Kah Mun, G., Hande, M.P., Valiyaveettil, S., 2009. Cytotoxicity and genotoxicity of silver nanoparticles in human cells. ACS nano. 3(2), 279-290.

-

Awadalla, H.N., El-Temsahi, M.M., Negm, A.Y., Abou El Naga, I.F., Zeid, M.H., 1995. Intestinal protozoal infections among HIV-infected individuals in Alexandria. J. Hepat. Gastroentrol. Inf. Dis. 4(1), 27-35.

-

Awwad, A.M., Salem, N.M., Abdeen, A.O., 2013. Green synthesis of silver nanoparticles using carob leaf extract and its antibacterial activity. Int. J. Ind. Chem. 4(1), 29.

-

Bayard, P.J., Berger, T.G., Jacobson, M.A., 1992. Drug hypersensitivity reactions and human immunodeficiency virus disease. J. Acquir. Immune Defic. Syndr. 5(12), 1237-1257.

Journal Pre-proof

-

Bednarska, M., Bajer, A., Welc-Falęciak, R., Pawełas, A., 2015.Cyclospora cayetanensis infection in transplant traveller: a case report of outbreak. Parasite Vectors 8:41.

-

Bernstein, L.S., 1975. Adverse reaction to trimethoprim-sulfamethoxazole, with particular reference to long-term therapy. Can. Med. Assoc. J. 112, 96-98.

-

Cameron, P., Gaiser, B.K., Bhandari, B., Bartley, P.M., Katzer, F., Bridle, H., 2015. Silver nanoparticles decrease the viability of Cryptosporidium parvum oocysts. Appl. Environ. Microbiol. 82(2), 431-437.

-

Chacin-Bonilla, L., 2010. Epidemiology of Cyclospora cayetanensis: A review focusing in endemic areas.Acta. Trop. 115(3), 181-193.

-

Chacin-Bonilla, L., 2012. Cyclospora cayetanensis as a Global Health Problem. Epidemiology 2, 3 http://dx.doi.org/10.4172/2161-1165.

-

Cho, K.H., Park, J.E., Osaka, T., Park, S.G., 2005. The study of antimicrobial activity and preservative effects of nanosilver ingredient. Electrochim. Acta 51(5), 956-960.

-

Chorawala, M.R., Trivedi, V.R., Dave, D.J., Oza, P.M., Shah, G.B., 2013. Acute and subacute toxicity studies of cell wall contents of probiotic (Lactobacillus casei) in Wistar rats and Swiss Albino mice. RJPBCS 4(2), 719-732.

-

Choudhry, N., Korbel, D.S., Edwards, L.A., Bajaj-Elliott, M., McDonald, V., 2009. Dysregulation of interferon-gamma-mediated signalling pathway in intestinal epithelial cells by Cryptosporidium parvum infection. Cell Microbiol. 11(9), 1354-1364.

-

Curry, A., Smith, H.V., 1998. Emerging pathogens: Isospora, Cyclospora and microsporidia. Parasitology 117, 143-159.

-

De Górgolas, M., Fortés, J., Fernández Guerrero, M.L., 2001.Cyclospora cayetanensis cholecystitis in a patient with AIDS. Ann. Intern. Med. 134(2), 166.

Journal Pre-proof

-

El Fakhry, Y., Achbarou, A., Desportes-Livage, I., Mazier, D., 1998. Encephalitozoon intestinalis: humoral responses in interferon-gamma receptor knockout mice infected with a microsporidium pathogenic in AIDS patients. Exp. Parasitol. 89(1), 113-121.

-

Fang, J., Zhong, C., Mu, R., 2005. The study of deposited silver particulate films by simple method for efficient SERS. Chemi. Physi. Lett. 401(1), 271-275.

-

Fraser, T.N., Avellaneda, A.A., Graviss, E.A., Musher, D.M., 2012. Acute kidney injury associated with trimethoprim/sulfamethoxazole. J. Antimicrob. Chemother.

67(5), 1271-

1277. -

Gaafar, M.R., Mady, R.F., Diab, R.G., Shalaby, T.I., 2014. Chitosan and silver nanoparticles: promising anti-toxoplasma agents. Exp. Parasitol. 143, 30-38.

-

Garcia, L.S., 2016. Intestinal Protozoa (Coccidia), microsporidia and Algae. In: Diagnostic Medical Parasitology, 6th edition, ASM press, Washington, D.C., PP. 612-666.

-

Gherbawy, Y.A., Shalaby, I.M., El-Sadek, M.S., Elhariry, H.M., Abdelilah, B.A., 2013. The anti-fasciolasis properties of silver nanoparticles produced by Trichoderma harzianum and their improvement of the anti-fasciolasis drug triclabendazole. Int. J. Mol. Sci.14(11), 2188721898.

-

Gleckman, R., Blagg, N., Joubert, D.W., 1981. Trimethoprim: mechanisms of action, antimicrobial activity, bacterial resistance, pharmacokinetics, adverse reactions, and therapeutic indications. Pharmacotherapy 1(1), 14-20.

-

Gong, J.L., Liang, Y., Huang, Y., Chen, J.W., Jiang, J.H., Shen, G.L., Yu, R.Q., 2007. Ag/SiO2 core-shell nanoparticle-based surface-enhanced Raman probes for immunoassay of cancer marker using silica-coated magnetic nanoparticles as separation tools. Biosens. Bioelectron. 22(7), 1501-1507.

Journal Pre-proof

-

Hands-chumacher, R.E., 1991. Drugs used for immunosuppression. Immunosuppressive agents. In: Gilman, AG. RT., Nies, A.S., Taylor, P.(Ed.) The Pharmacological Basis of Therapeutics, 8thed., Vol II, Maxwell Macmillan Pergamon Publishing Corporation, PP. 12641276.

-

Kalkut, G., 1998. Sulfonamides and trimethoprim. Cancer Invest. 16(8), 612-615.

-

Khalifa, A.M., El-Temsahy, M.M., Abou El Naga, I.F., 2001. Effect of ozone on the viability of some protozoa in drinking water. J. Egypt. Soc. Parasitol. 31(2), 603-616.

-

Kirkpatrick, L.A., Feeney, B.C., 2013. A simple guide to IBM SPSS: for version 20.0. student Ed. Belmont, Calif, Wadsworth, Cengage Learning.

-

Klainer, A.S., Betsch, C.J., 1970. Scanning-beam electron microscopy of selected microorganisms. J. Infect. Dis. 121 (3), 339–343.

-

Kotz, S., Balakrishnan, N., Read, C., Vidakovic, B., 2006. Encyclopedia of statistical sciences, 2nd ed., Wiley Interscience Hoboken, NJ.

-

Kowdley, K.V., Keeffe, E.B., Fawaz, K.A., 1992. Prolonged cholestasis due to trimethoprim sulfamethoxazole. Gastroenterology 102(6), 2148-2150.

-

Lacroix-Lamande, S., Mancassola, R., Naciri, M., Laurent, F., 2002. Role of gamma interferon in chemokine expression in the ileum of mice and in a murine intestinal epithelial cell line after Cryptosporidium parvum infection. Infect. Immun.70(4), 2090-2099.

-

Lee, M., Bozzo, P., Einarson, A., Koren, G., 2008. Urinary tract infections in pregnancy. Can. Fam. Physician 54(6), 853-854.

-

Loeschner, K., Hadrup, N., Qvortrup, K., Larsen, A., Gao, X., Vogel, U.,Mortensen, A., Lam, H.R., Larsen, E.H., 2011. Distribution of silver in rats following 28 days of repeated oral exposure to silver nanoparticles or silver acetate. Part. Fibre Toxicol. 8, 18.

Journal Pre-proof

-

MacMicking, J.D., 2012. Interferon-inducible effector mechanisms in cell-autonomous immunity. Nat. Rev. Immunol. 12(5), 367-382.

-

Madico, G., McDonald, J., Gilman, R.H., Cabrera, L., Sterling, C.R., 1997. Epidemiology and treatment of Cyclospora cayetanensis infection in Peruvian children. Clin. Infect. Dis. 24(5), 977-981.

-

Malaikozhundan, B., Vaseeharana, B., Vijayakumara, S., Sudhakaranb, R., Gobia, N., Shanthinic, G., 2016. Antibacterial and antibiofilm assessment of Momordicacharantia fruit extract coated silver nanoparticle. Biocatal. Agric. Biotechnol. 8, 189-196.

-

Marinac, J.S., Stanford, J.F., 1993. A severe hypersensitive reaction to trimethoprimsulfamethoxazole in a patient infected with human immunodeficiency virus. Clin. Infect. Dis. 16(1), 178-179.

-

Martine,

D.,

1989.

Antineoplastic

agents

and

immunosuppressants.

In:

The

Etropharmacopecia, 2nded.,Pharmacutical Press London, PP. 580-612. -

Midha, K., Singh, G., Nagpal, M., Arora, S., 2016. Potential application of silver nanoparticles in medicine. Nanoscience & Nanotechnology-Asia 6(2), 82-91.

-

Mukherjee, S., Chowdhury, D., Kotcherlakota, R., Patra, S.B. V., Bhadra, M.P.,Sreedhar, B., Patra, C.R., 2014. Potential theranostics application of bio-synthesized silver nanoparticles (4in-1 system). Theranostics 4(3), 316-335.

-

Murphy, A., Casey, A., Byrne, G., Chambers, G., Howe, O., 2016. Silver nanoparticles induce pro-inflammatory gene expression and inflammasome activation in human monocytes. J. Appl. Toxicol. 36(10), 1311-1320.

-

Neena, D., Devraj, K.M., Bhagat, A.P., 2012. Role of capping agent in the synthesis of silver nanoparticles. J. Pharmacy Res. 5(9),4710-4712.

Journal Pre-proof

-

Nooshin, K.D., 2016. Silver nanoparticles as a novel approach to the treatment of chronic lyme disease and associated diseases. Towns end Letter 48-57.

-

Norouzi, R., 2017. A review on most nanoparticles applied against parasitic infections. J. Biol. and Today's World 6(10), 196-203.

-

Ognik, K., Cholewińska, E., Czech, A., Kozłowski, K., Wlazło, Ł., Nowakowicz-Dębek, B., Szlązak, R., Tutaj, K., 2016. Effect of silver nanoparticles on the immune, redox, and lipid status of chicken blood K. Czech. J. Anim. Sci. 61(10), 450–461.

-

Ognik, K., Sembratowicz, I., Cholewińska, E., Wlazło, Ł., Nowakowicz-Dębek, B., Szlązak, R., Tutaj, k., 2016. The effect of chemically-synthesized silver nanoparticles on performance and the histology and microbiological profile of the jejunum in chickens. Ann. Anim. Sci. 16(2), 439-450.

-

Oliveira, M., Ugarte, D., Zanchet, D., Zarbin A., 2005. Influence of synthetic parameters on the size, structure, and stability of dodecanethiol-stabilized silver nanoparticles. J. Colloid Interface Sci. 292, 429-435.

-

Ortega, Y.R., Sanchez, R., 2010. Update on Cyclospora cayetanensis, a food-borne and waterborne parasite. Clin. Microbiol. Rev. 23(1), 218-234.

-

Pape, J.W., Verdier, R.I., Bondy, M., Boncy, J., Johnson, W.D., 1994. Cyclospora infection in adults infected with HIV. Clinical manifestations, treatment, and prophylaxis. Ann. Intern. Med. 121(9), 654-657.

-

Penido, M.L.O., Nelson, D.L., Vieira, L.Q., Coelho, P.M.Z., 1994. Schistosomicidal activity of alkylaminooctanethiosulfuric acids. Mem. Inst. Oswaldo Cruz 89(4), 595-602.

Journal Pre-proof

-

Pollok, R.C., Farthing, M.J., Bajaj-Elliott, M., Sanderson, I.R., McDonald, V., 2001. Interferon gamma induces enterocyte resistance against infection by the intracellular pathogen Cryptosporidium parvum. Gastroenterology 120(1), 99-107.

-

Ponarulselvam, S., Panneerselvam, C., Murugan, K., Aarthi, N., Kalimuthu, K., Thangamani, S., 2012. Synthesis of silver nanoparticles using leaves of Catharanthus roseus Linn. G. Don and their antiplasmodial activities. Asian Pac. J. Trop. Biomed. 2(7), 574-580.

-

Prabhu, S., Poulose, E.K., 2012. Silver nanoparticles: mechanism of antimicrobial action, synthesis, medical applications, and toxicity effects. Int. Nano. Lett. 2 (1), 32-41.

-

Saad, H.A., Soliman, M.I., Azzam, A.M., Mostafa, A.B., 2015. Antiparasitic activity of silver and copper oxide nanoparticles against Entamoeba histolytica and Cryptosporidium parvum cysts. J. Egypt. Soc. Parasitol.45(3), 593-602.

-

Sabella, S., Carney, R.P., Brunetti, V., Malvindi, M.A., Al-Juffali, N., Vecchio, G.,Janes, S.M., Bakr, O.M., Cingolani, R., Stellacci, F., Pompa, P.P., 2014. A general mechanism for intracellular toxicity of metal-containing nanoparticles. Nanoscale 6(12), 7052-7061.

-

Said, D.E., El Samad, L.M., Gohar, Y.M., 2012. Validity of silver, chitosan, and curcumin nanoparticles as anti-Giardia agents. Parasitol. Res. 111(2), 545–554.

-

Sankar, R., Karthik, A., Prabu, A., Karthik, S., Shivashangari, K.S., Ravikumar, V., 2013. Origanum vulgare mediated biosynthesis of silver nanoparticles for its antibacterial and anticancer activity. Colloids and Surf B Biointerfaces 108, 80-84.

-

Sondi, I., Salopek-Sondi, B., 2004. Silver nanoparticles as antimicrobial agent: a case study on E. coli as a model for Gram-negative bacteria. J. Colloid Interface Sci. 275(1), 177-182.

-

Sherwood, D., Angus, K.W., Snodgrass, D.R., Tzipori, S., 1982. Experimental cryptosporidiosis in laboratory mice. Infect. Immun. 38(2), 471-475.

Journal Pre-proof

-

Smith, E., Light, J., Filo, R., Yum, M.N., 1980. Interstitial nephritis caused by trimethoprimsulfamethoxazole in renal transplant recipients. JAMA 244(4), 360-361.

-

Smith, H.V., Paton, C.A., Mitambo, M.M., Girdwood, R.W., 1997. Sporulation of Cyclospora sp. oocysts. Appl. Environ. Microbiol. 63(4), 1631-1632.

-

Steenbergen, J., Shuman, H., Casadevall, A., 2001. Cryptococcus neoformans interactions with amoebae suggest an explanation for its virulence and intracellular pathogenic strategy in macrophages. Proc. Nalt. Acad. Sci. 98(26), 15245-15250.

-

Steinberg, A.D., Steinberg, S.C., 1991. Long-term preservation of renal function in patients with lupus nephritis receiving treatment that includes cyclophosphamide versus those treated with prednisone only. Arthritis Rheum. 34(8), 945-950.

-

Sturge, C.R., Benson, A., Raetz, M., Wilhelm, C.L., Mirpuri, J., Vitetta, E.S., Yarovinsky, F., 2013. TLR-independent neutrophil-derived IFN-γ is important for host resistance to intracellular pathogens. Proc. Natl. Acad. Sci.110(26), 10711-10716.

-

Subramaniam, S.R., Cader, R.A., Mohd, R., Yen, K.W., Ghafor, H.A., 2013. Low-dose cyclophosphamide-induced acute hepatotoxicity. Am. J. Case Rep. 14, 345-349.

-

Szczepanowicz, K., Stefanska, J., Socha, R.P., Warszynski, P., 2010. Preparation of silver nanoparticles via chemical reduction and their antimicrobial activity. Physicochem. Probl. Miner Process 45, 85-98.

-

Turchany, J.M., Aley, S.B., Gillin, F.D., 1995. Giardicidal activity of lactoferrin and Nterminal peptides. Infect. Immun. 63(11), 4550-4552.

-

Verdier, R.I., Fitzgerald, D.W., Johnson, W.D., Pape, J.W., 2000. Trimethoprimsulfamethoxazole compared with ciprofloxacin for treatment and prophylaxis of Isospora belli

Journal Pre-proof

and Cyclospora cayetanensis infection in HIV-infected patients. A randomized, controlled trial. Ann. Intern. Med. 132(11), 885-888. -

Vijayakumar, S., Malaikozhundan, B., Ramasamy, P., Vaseeharan, B., 2016. Assessment of biopolymer stabilized silver nanoparticle for their ecotoxicity on Ceriodaphniacornuta and antibiofilm activity. J. Environ. Chem. Eng. 2, 2076-2083.

-

Wanat, K.A., Anadkat, M.J., Klekotka, P.A., 2009. Seasonal variation of Stevens-Johnson syndrome and toxic epidermal necrolysis associated with trimethoprim-sulfamethoxazole. J. Am. Acad. Dermatol. 60(4), 589-594.

-

Xu, Y., Tang, H., Liu, J.H., Wang, H., Liu, Y., 2013. Evaluation of the adjuvant effect of silver nanoparticles both in vitro and in vivo. Toxicol. Lett. 219(1), 42-48.

-

Yasuoka, A., Oka, S., Komuro, K., Shimizu, H., Kitada, K., Nakamura, Y., Shibahara, S., Takeuchi, T., Kondo, S., Shimada, K., 1995. Successful treatment of Pneumocystis carinii pneumonia in mice with benanomicin A (ME1451). Antimicrob. Agents Chemother. 39(3), 720-724.

-

Yousef, J., Hendi, H., Hakami, F.S., Awad, M.A., Alem, A.F., Hendi, A.A., 2012. Toxicity of silver nanoparticles after injected intraperitoneally in rats. J. Am. Sci. 8(3), 589-593.

-

Yun, C.H., Lillehoj, H.S., Zhu, J., Min, W., 2000. Kinetic differences in intestinal and systemic interferon-gamma and antigen-specific antibodies in chickens experimentally infected with Eimeria maxima. Avian Dis. 44(2), 305-312.

-

Zar, F.A., El-Bayoumi, E., Yungbluth, M.M., 2001. Histologic proof of acalculous cholecystitis due to Cyclospora cayetanensis. Clin. Infect. Dis. 33(12), 140-141.

Journal Pre-proof

-

Zhang, X.F., Liu, Z.G., Shen, W., Gurunathan, S., 2016. Silver Nanoparticles: Synthesis, characterization, properties, applications, and therapeutic approaches. Int.J. Mol. Sci. 17(9), 134.

-

Zoheir, K.M., Harisa, G.I., Abo-Salem, O.M., Ahmad, S.F., 2015. Honey bee is a potential antioxidant against cyclophosphamide-induced genotoxicity in albino male mice. Pak. J. Pharm. Sci. 28(3), 973-981.

-

Cyclosporiasis Outbreak Investigations - United States, 2017 [cited 2018 June]. Available from: https:/www.cdc.gov/parasites/cyclosporiasis/outbreaks/2017/index.html.

Legends of figures Fig. 1: The change in colour during preparation of Ag NPs (a) colourless solution before Ag NPs formation (b) pale yellow solution indicating Ag NPs formation. Fig. 2: TEM image and size distribution histogram of Ag NPs (x25000). Fig. 3: Results of Ag NPs characterization: (a) UV-Visible spectrum of the Ag NPs. (b) Fourier transform infra-red spectra of Ag NPs. (c) Particle size analysis of the prepared Ag NPs. (d) Zeta potential of the prepared Ag NPs. Fig. 4: Cyclospora oocysts in patient’s stool stained with safranin stain (×1000). Fig. 5: (a) Viable Cyclospora oocysts, and (b) Dead Cyclospora oocysts in mice stool stained by trypan blue stain (×400).

Journal Pre-proof

Plate I: SEM of C. cayetanensis oocysts in stool of infected non-treated, co-trimoxazoletreated and Ag NPs-treated mice. Fig. a: normal non-treated spherical oocysts with outer smooth regular fibrillar coats and an indentation on the surface at one side (x35000). Fig. b: co-trimoxazole-treated shrunken oocysts of subgroup IIA1 with multiple compressions and pores (x35000). Fig. c: co-trimoxazole-treated oocysts of subgroup IIB1 demonstrating irregularities on their surface with pores, papules and dimples (x35000). Fig. d: Ag NPstreated cauliflower-like oocysts of subgroup IIA2 with evident deep perforations throughout their diameters (x35000). Fig. e: Ag NPs-treated oocysts of subgroup IIA2 revealing deep clefts and furrows in their surfaces (x35000). Fig. f: Ag NPs-treated shrunken oocysts of subgroup IIB2 with obvious deep pores and cavitations (x35000).

Journal Pre-proof

-

Silver nanoparticles proved their effectiveness against experimental Cyclospora infection.

-

Animals that received silver nanoparticles showed statistically significant decrease in the oocyst burden and number of viable oocysts in stool.

-

A statistically significant increase in serum IFN-γ was recorded in the silver nanoparticles treated group.

-

SEM revealed irregularities, poring and perforations in the treated Cyclospora oocysts.

-

No evidence of toxicity of silver nanoparticles.

Table (I): Comparison between the oocyst count in stool of both immunocompetent infected subgroups treated with cotrimoxazole (IIA1) and Ag NPs (IIA2) and their corresponding infected non-treated subgroup (IB1) at the different durations of examination. 6th day Min. – Max. x ± SD. Median % reduction 8th day Min. – Max. x ± SD. Median % reduction 10th day Min. – Max. x ± SD. Median % reduction 12th day Min. – Max. x ± SD. Median % reduction 14th day Min. – Max. x ± SD. Median % reduction

Subgroup IB1

Subgroup IIA1

Subgroup IIA2

0.0 – 3.0 1.50 ± 1.18 1.0

0.0 – 3.0 1.40 ± 1.07 1.0 %R1=6.7

0.0 – 3.0 1.20 ± 1.14 1.0 %R2=20

2.0 - 6.0 4.0 ± 1.41 3.50

0.0 – 5 3.50 ± 1.58 4.0 %R1=12.5

1.0 – 5.0 2.90 ± 1.45 3.0 %R2=27.5

2.0 – 9.0 6.0 ± 1.94 6.0

0.0 – 4.0 2.70 ± 1.34 3.0 %R1=55

1.0 – 5.0 2.30 ± 1.42 2.0 %R2=61.7

3.0 – 9.0 6.30 ± 1.89 6.50

0.0 – 4.0 1.70 ± 1.42 2.0 %R1=73

0.0 – 3.0 1.10 ± 0.99 1.0 %R2=82.5

3.0 – 9.0 6.50 ± 1.90 6.50

0.0 – 3.0 1.10 ± 1.10 1.0 %R1=83.1

0.0 – 1.0 0.20 ± 0.42 0.0 %R2=96.9

p1

p2

p3

0.874

0.551

0.635

0.642

0.111

0.297

0.001*

0.001*

0.415

<0.001*

<0.001*

0.330

<0.001*

<0.001*

0.039*

p1: p value for comparing between IB1and IIA1 p2: p value for comparing between IB1and IIA2 p3: p value for comparing between IIA1and IIA2 *: Statistically significant at p ≤ 0.05 using Mann Whitney test. %R1: % reduction in the oocyst count in stool of IIA1 in relation to IB1 %R2: % reduction in the oocyst count in stool of IIA2 in relation IB1

Table (II): Comparison between the oocyst count in stool of both immunosuppressed infected subgroups treated with co-trimoxazole (IIB1) and Ag NPs (IIB2) and their corresponding infected non-treated subgroup (IB2) at the different durations of examination. Subgroup IB2 day Min. – Max. x ± SD. Median % reduction th 8 day Min. – Max. x ± SD. Median % reduction 10th day Min. – Max. x ± SD. Median % reduction 12th day Min. – Max. x ± SD. Median % reduction 14th day Min. – Max. x ± SD. Median % reduction

Subgroup IIB1

Subgroup IIB2

2.0 – 6.0 3.50 ± 1.27 3.0

1.0 – 5.0 2.90 ± 1.1 3.0 %R1=17.1

1.0 – 4.0 2.70 ± 1.06 2.5 %R2=22.9

3.0 – 7.0 5.30 ± 1.25 5.5

2.0 – 6.0 4.0 ± 1.25 4.0 %R1=24.5

0.0 – 6.0 3.50 ± 1.78 4.0 %R2=33.9

5.0 – 10.0 7.0 ± 0.82 7.0

2.0 – 6.0 3.5 ± 1.15 3.0 %R1=50

0.0 – 5.0 2.10 ± 1.45 2.0 %R2=70

6.0 – 11.0 8.0 ± 0.82 8.0

0.0 – 4.0 2.40 ± 1.43 3.0 %R1=70

0.0 – 3.0 1.0 ± 1.15 0.50 %R2=87.5

7.0 – 12.0 9.20 ± 1.08 9.50

0.0 – 3.0 1.70 ± 1.16 2.0 %R1=81.5

0.0 – 2.0 0.70 ± 0.82 0.50 %R2=92.4

p1

p2

p3

0.128

0.183

0.694

0.200

0.103

0.589

0.001*

<0.001*

0.007*

<0.001*

<0.001*

0.028*

<0.001*

<0.001*

0.049*

6th

p1: p value for comparing between IB2 and IIB1

p2: p value for comparing between IB2 and IIB2

p3: p value for comparing between IIB1 and IIB2

*: Statistically significant at p ≤ 0.05 using Mann Whitney test. %R1: % reduction in the oocyst count in stool of IIB1 in relation to IB2

%R2: % reduction in the oocysts count in stool of IIB2 in relation to IB2

Table (III): Comparison between the oocysts viability in stool of all infected subgroups on the 14th day P.I. Infected immunocompetent subgroups

Infected immunosuppressed subgroups

Infected treated subgroups Infected non(IIA) treated Co-trimoxazole Ag NPs treated subgroup treated (IB 1)

subgroup

subgroup (IIA2)

(IIA1) Viable oocysts Min. – Max. x̅ ± SD. Median p1/p4

3.0 – 9.0 6.0 ± 1.25 6.0

0.0 – 2.0 0.80 ± 0.79 1.0

subgroup (IB2) 7.0 – 11.0 8.8 ± 1.41 8.5

p1 <0.001* p2 <0.001*

Co-trimoxazole Ag NPs treated treated subgroup subgroup (IIB1) (IIB2) 0.0 – 3.0 1.50 ± 1.18 2.0

0.0 – 0.0 0.0 ± 0.0 0.0 p5 <0.001*

p3 = 0.005* 92.3

Infected treated subgroups (IIB)

p4 <0.001*

p2/p5 p3/p6 % reduction % of Viability

0.0 – 0.0 0.0 ± 0.0 0.0

Infected nontreated

%R1=86.7 72.7

%R2=100 0.0

p1: p value for comparing between IB1and IIA1 p2: p value for comparing between IB1and IIA2 p3: p value for comparing between IIA1and IIA2

95.6

p6 = 0.002* %R3=82.9 %R4=100 88.2 0.0

p4: p value for comparing between IB2 and IIB1 p5: p value comparing between IB2 and IIB2 p6: p value comparing between IIB1 and IIB2

*: Statistically significant at p ≤ 0.05 using Mann Whitney test %R1: % reduction in the number of viable oocysts in IIA1 in relation to IB1 %R2: % reduction in the number of viable oocysts in IIA2 in relation to IB1

%R3: % reduction in the number of viable oocysts in IIB1 in relation to IB2 %R4: % reduction in the number of viable oocysts in IIB2 in relation to IB2

Table (IV): Comparison between the level of interferon gamma (IFN-γ) in sera of all studied subgroups on the 14th day P.I. IFN-γ Sub-groups

x ± SD. (pg/ml)

Significance between Subgroups

IA1 39.0 ± 5.44 IC1a 38.0 ± 2.79 p1=1.000 IC1b 130.0 ± 3.71 p2<0.001* p3<0.001* IB1 235.0 ± 3.80 p4<0.001* IIA1 238.0 ± 3.94 p5=0.806 IIA2 485.0 ± 2.94 p6<0.001* p7<0.001* IA1: Non-infected non-treated immunocompetent subgroup IC1a: Non-infected co-trimoxazole-treated immunocompetent subgroup IC1b: Non-infected Ag NPs-treated immunocompetent subgroup IB1: Infected non-treated immunocompetent subgroup IIA1: Infected co-trimoxazole-treated immunocompetent subgroup IIA2: Infected Ag NPs-treated immunocompetent subgroup

IFN-γ Sub-groups

x ± SD. (pg/ml)

Significance between Subgroups

IA2 ND IC2a ND IC2b 42.0 ± 5.16 IB2 45.0 ± 4.08 IIB1 46.0 ± 2.55 p8=1.000 IIB2 155.0 ± 4.55 p9<0.001* p10<0.001* IA2:Non-infected non-treated immunosuppressed subgroup IC2a: Non-infected co-trimoxazole-treated immunosuppressed subgroup IC2b: Non-infected Ag NPs-treated immunosuppressed subgroup IB2: Infected non-treated immunosuppressed subgroup IIB1: Infected co-trimoxazole-treated immunosuppressed subgroup IIB2: Infected Ag NPs-treated immunosuppressed subgroup

p1: p value between IA1 & IC1a p2: p value between IA1 & IC1b p3: p value between IC1a & IC1b p4: p value between IA1& IB1 p5: p value between IB1 & IIA1

p6: p value between IB1 & IIA2 p7: p value between IIA1 & IIA2 p8: p value between IB2 & IIB1 p9: p value between IB2 & IIB2 p10: p value between IIB1 & IIB2

*: Statistically significant at p ≤0.05 using PostHoc Test (Tukey)

ND: Non-detectable

Table (V): Comparison between the level of liver aspartate transaminase (AST) and liver alanine transaminase (ALT) in sera of all studied subgroups on the 14th day P.I. Liver enzymes Sub-

AST (SGOT) x̅ ± SD. (IU/L)

groups

Immunocompetent Subgroups

Immunosuppressed Subgroups

ALT (SGPT)

Significance between Subgroups

IA1

23.0 ± 5.31

IC1a

21.50 ± 5.23

p1=1.000

IC1b

13.0 ± 3.80

p2=0.992

IB1

16.90 ± 1.58

IIA1

17.0 ± 2.05

p4=1.000

IIA2

15.20 ± 2.34

p5=1.000

IA2

69.0 ± 25.58

IC2a

85.0 ± 39.51

p7=0.808

IC2b

20.10 ± 4.01

p8<0.001*

IB2

76.0 ± 27.16

IIB1

83.0 ± 39.94

p10=1.000

IIB2

17.0 ± 5.87

p11<0.001*

x̅ ± SD. (IU/L)

Significance between Subgroups

26.0 ± 2.18 p3=0.998

23.0 ± 2.47

p1=0.945

22.0 ± 4.18

p2=1.000

p3=0.945

25.0 ± 5.91 p6=1.000

26.0 ± 3.80

p4=0.833

24.0 ± 3.50

p5=0.833

p6=1.000

78.05 ± 2.96 p9<0.001*

80.0 ± 30.64

p7= 1.000

14.80 ± 2.93

p8<0.001*

p9<0.001*

79.0 ± 26.85

IA1: Non-infected non-treated immunocompetent subgroup IC1a: Non-infected co-trimoxazole-treated immunocompetent subgroup IC1b: Non-infected Ag NPs-treated immunocompetent subgroup IB1: Infected non-treated immunocompetent subgroup IIA1: Infected co-trimoxazole-treated immunocompetent subgroup IIA2: Infected Ag NPs-treated immunocompetent subgroup

p12<0.001*

80.0 ± 30.07

p10= 1.000

25.0 ± 4.76

p11<0.001*

p12<0.001*

IA2:Non-infected non-treated immunosuppressed subgroup IC2a: Non-infected co-trimoxazole-treated immunosuppressed subgroup IC2b: Non-infected Ag NPs-treated immunosuppressed subgroup IB2: Infected non-treated immunosuppressed subgroup IIB1: Infected co-trimoxazole-treated immunosuppressed subgroup IIB2: Infected Ag NPs-treated immunosuppressed subgroup

p1: p value between IA1 & IC1a p2: p value between IA1 & IC1b p3: p value between IC1a & IC1b

p4: p value between IB1 & IIA1 p5: p value between IB1 & IIA2 p6: p value between IIA1 & IIA2

*: Statistically significant at p ≤ 0.05 using Post Hoc Test (Tukey)

p7: p value between IA2 & IC2a p8: p value between IA2 & IC2b p9: p value between IC2a & IC2b

p10: p value between IB2 & IIB1 p11: p value between IB2 & IIB2 p12: p value between IIB1 & IIB2

Table (VI): Comparison between the level of urea and creatinine in sera of all studied subgroups on the 14th day P.I. Urea / creatinine Sub-

Serum urea x̅ ± SD. (IU/L)

groups

Immunocompetent Subgroups

Immunosuppressed Subgroups

Significance between Subgroups

IA1

16.0 ± 1.90

IC1a

14.90 ± 4.13

p1=1.000

IC1b

13.60 ± 3.83

p2=1.000

IB1

15.50 ± 2.22

IIA1

12.90 ± 3.13

p4=0.985

IIA2

12.0 ± 6.10

p5=0.999

IA2

16.05 ± 1.34

IC2a

13.0 ± 8.10

p7<0.11

IC2b

12.10 ± 4.01

p8= 0.630

IB2

14.0 ± 9.76

IIB1

13.0 ± 8.10

p10=0.081

IIB2

12.0 ± 5.37

p11=0.376

Significance between Subgroups

p3=1.000

0.61 ± 0.15

p1=0.992

0.59 ± 0.19

p2=0.997

p3=1.000

0.80 ± 0.45 p6=1.000

0.76 ± 0.09

p4=0.071

0.51 ± 0.38

p5=0.785

p6=0.968

0.90 ± 0.47 p9<0.21

0.61 ± 0.20

p7= 1.000

0.53 ± 0.21

p8=0.071

p9=0.82

0.80 ± 0.78

p4: p value between IB1 & IIA1 p5: p value between IB1 & IIA2 p6: p value between IIA1 & IIA2

*: Statistically significant at p ≤ 0.05 using Post Hoc Test (Tukey)

x̅ ± SD. (IU/L)

0.80 ± 0.29

IA1: Non-infected non-treated immunocompetent subgroup IC1a: Non-infected co-trimoxazole-treated immunocompetent subgroup IC1b: Non-infected Ag NPs-treated immunocompetent subgroup IB1: Infected non-treated immunocompetent subgroup IIA1: Infected co-trimoxazole-treated immunocompetent subgroup IIA2: Infected Ag NPs-treated immunocompetent subgroup p1: p value between IA1 & IC1a p2: p value between IA1 & IC1b p3: p value between IC1a & IC1b

Serum creatinine

p12<0.15

0.71 ± 0.36

p10= 1.000

0.61 ± 0.50

p11=0.256

p12=0.568

IA2:Non-infected non-treated immunosuppressed subgroup IC2a: Non-infected co-trimoxazole-treated immunosuppressed subgroup IC2b: Non-infected Ag NPs-treated immunosuppressed subgroup IB2: Infected non-treated immunosuppressed subgroup IIB1: Infected co-trimoxazole-treated immunosuppressed subgroup IIB2: Infected Ag NPs-treated immunosuppressed subgroup p7: p value between IA2 & IC2a p8: p value between IA2 & IC2b p9: p value between IC2a & IC2b

p10: p value between IB2 & IIB1 p11: p value between IB2 & IIB2 p12: p value between IIB1 & IIB2