Evaluation of potential prophylactic and therapeutic effect of azoximer bromide (polyoxidonium) on experimental cryptosporidiosis in immunocompromised mice

Background: Considering the broad burden of cryptosporidiosis, there is still a limited choice of curative treatments. Nitazoxanide (NTZ) is the only anti-cryptosporidial agent currently available. Unfortunately, it showed low efficacy in children and AIDS patients. Accordingly, supplementation with immune-stimulation drugs is feasible. Objective: To demonstrate the prophylactic immunomodulating effect of the immunostimulant Azoximer Bromide (AZB) and evaluate its potential therapeutic efficacy when combined with NTZ, for treatment of cryptosporidiosis in experimentally immunosuppressed mice. Material and Methods: Ninety laboratory bred Swiss albino male mice were immunosuppressed and divided into three groups (30 mice each): control group (GI); prophylactic group, AZB treated then infected (GII); therapeutic group, oocysts infected then treated (GIII). Each group was divided equally into 3 sub-groups (10 mice each). Controls included: GIa, non-infected control negative; GIb, oocysts infected control positive; GIc, non-infected AZB treated drug control. Prophylactic subgroups included: GIIa, received AZB booster injection; GIIb, NTZ treated; GIIc, AZB+ NTZ treated. Therapeutic subgroups included: GIIIa, AZB treated; GIIIb, NTZ treated; GIIIc, AZB+NTZ treated. Oocysts shedding and the efficacy percentage of each drug were calculated. Other parameters used included histopathological examination and immunohistochemical assessment of small intestine and lung tissues, and serum analyses for biochemical, immunological and antioxidants evaluations. Results: The prophylactic effect of AZB alone and its therapeutic effect when combined with NTZ gave the best reduction rate of oocyst shedding with marked improvement in histopathological features, and significantly reduced hepatic enzymes. Additionally, AZB enhanced the mice immunogenicity with significant upregulation of interleukin (IL)-1β, IL-6, tumor necrotic factor (TNF)-α and interferon (INF)-γ; overexpression of CD3 protein in pulmonary tissue, and significant elevation of antioxidant activity. Conclusion: A powerful effect was achieved by AZB when administered with NTZ for treatment of experimental cryptosporidiosis with elicited high immune response of immunosuppressed mice. PARASITOLOGISTS UNITED JOURNAL 294 and plays an important role in both innate and adaptive immune responses[8]. In the acute phase of infection, Cryptosporidium spp. sporozoites induce the production of IL-12 by macrophages and dendritic cells[9] that acts synergistically with IL-18 and TNF-α to activate natural killer (NK) cells[10]. In addition, TNF-α prevents the establishment of Cryptosporidium spp. infection in enterocytes[11]. Besides, other proinflammatory cytokines (IL-1, IL-6) released by multiple immunocompetent cells exert protective effect[12]. Adequate T helper cell responses are critical for hosts to orchestrate enough defensive mechanisms for infection control. This suggests a major role for host immune factors in controlling cryptosporidiosis[13]. Oxidative stress induced by Cryptosporidium spp. was reported to cause tissue damage in mice[14] and pigs[15]. So, decreasing oxidative stress allows the host to sustain a viable immune assemblage able to eradicate the pathogen and reduce host tissue damage[16]. Alterations in total antioxidant capacity (TAC) and malondialdehyde (MDA) concentration are valuable biomarkers to evaluate oxidative stress[17]. Currently approved therapeutics, NTZ and paromomycin, have limited activity in immunocompromised individuals[18]. Several drugs and drug combinations such as rifaximin and azithromycin were also investigated against cryptosporidiosis, with unsatisfactory results[19]. Limited treatment options create an urgent need for the development of new antiparasitic drugs. For this purpose the additional use of non-specific immunostimulator drugs is a reasonable option to strengthen the body's resistance to parasitic infection[20]. Azoximer bromide (Polyoxidonium®) is a physiologically active compound from a new class of heterochain aliphatic polyamines. It is a highmolecular-weight synthetic immune modulator drug that increases the resistance to local and general infection and is indicated for the treatment of viral infections[21]. It is worth mentioning that AZB is approved in Russia as a vaccine adjuvant drug that stimulates antibody production. According to an analysis of about 50 million recipients, AZB complexed with antigen in a commercial influenza vaccine demonstrated high safety[22]. Furthermore, it is used in various conditions that include bronchial asthma[23], chronic recurrent herpes simplex infections[24], pneumonia[25], pyelonephritis[26], recurrent urogenital chlamydial infections[27] and atopic dermatitis[28]. The professed immunomodulatory action of AZB elicits or amplifies an immune response in immunosuppressed patients[29], and is acknowledged as an immune modulator for the treatment of parasitic diseases[21]. Difficulties in controlling unfavorable consequences of cryptosporidial diarrhea in immunodeficient individuals prompted us to consider the probable protective and/or curative value of AZB versus NTZ. Using AZB may prohibit the fulminant outcomes and/ or ameliorate the immune response in Cryptosporidium infected immunocompromised hosts. Our present work aimed to evaluate the prophylactic and therapeutic efficacy of immunostimulant AZB combined with NTZ, and their dual role in experimentally immunosuppressed mice exposed to cryptosporidiosis. MATERIAL AND METHODS This experimental case-control study began in April 2019 and was completed in May 2020. It was conducted at the Laboratories of the Medical Parasitology and Pathology Departments, Faculty of Medicine, Menoufia University. Experimental animals: This study was performed on 90 Swiss albino male laboratory-bred mice weighing 20±3 gm. Mice were obtained from Schistosoma Biological Supply Program (SBSP), Theodor Bilharze Research Institute (TBRI), Giza, Egypt and kept under standard housing conditions in the animal house of TBRI. The mice were allowed to adapt to the experimental conditions for 10 days before Cryptosporidium infection. Mice were kept in separate cages under optimum conditions[30]. Private laboratory mouse pellets as food and water were also readily accessible. During this period, stool examination of all mice was conducted to ensure that they were parasites free. Study design: Ninety mice were immunosuppressed for 14 days and then classified into three main groups (I, II, III). Each group was divided equally into three subgroups (a, b, c), consisting of 10 mice each (Table 1). All the survived mice were sacrificed on the 30th day post infection (dpi) by cervical dislocation[32]. The effect of the drugs on murine cryptosporidiosis was evaluated by parasitological, histopathological, immunohistochemical, and immunological examinations. Mice immunosuppression: All mice were immunosuppressed by oral administration of synthetic corticosteroid (Dexazone tablets 0.5 mg, Al Kahira Pharmaceutical, and Chemical Industries Company, Egypt) at a dose of 25 μg/gm body weight/d for 14 successive days before oral inoculation with Cryptosporidium spp. oocysts[33,34]. Mice infection: Cryptosporidium oocysts collected from the feces of naturally infected calves[35] were identified by Modified Zeihl Neelsen (MZN) staining[36]. Oocysts were concentrated by floatation in Sheather’s sugar solution and the sediment was collected and stored in a 2.5% potassium dichromate solution at 4°C[37]. Before infection, oocysts were concentrated and counted in Azoximer bromide in experimental cryptosporidiosis Atia et al., 295 PBS solution using a hemocytometer. The mice were infected intra-esophageally with 30003500 oocysts using a tuberculin syringe[38]. All groups were observed daily for recording the mortality rate throughout the experimental period. Drug regimen: NTZ was supplied as “Cryptonaz®” 60 ml suspension of 100 mg/5 ml by Copad Pharma (Egypt for Trade and Pharmaceutical industries, Obour City, Cairo, Egypt). It was given orally to mice of subgroups II b, II c, III b, and III c at a dose of 500 mg/kg twice daily starting on the 15th dpi for five consecutive days[32]. The dose was calculated according to the Paget and Barnes table[33]. AZB was supplied as "Polyoxidonium®" 6 mg lyophilisate for preparation of solution for injection by Cosmic Nootropic Russien company. It was given to the prophylactic groups by IM injection twice in a dose of 0.004 mg/mouse in 0.2 ml 0.9% NaCl with an interval of 48 h, and given to therapeutic groups (subgroup IIIa and IIIc) in the same dose starting on the 15th dpi[31]. Parasitological evaluation of the infection: On the last day of the experiment (30th dpi) fresh fecal pellets were collected from each mouse separately and labeled individually for oocyst count and examined by the MZN staining method to calculate Cryptosporidium spp. oocysts shedding[32]. The smears were examined using a laboratory microscope and examined by x40 and x100 objectives. The number of Cryptosporidium spp. oocysts was counted and estimated as the mean in 10 high power fields (HPFs)[33]. The reduction percent of each drug was calculated using the equation: Efficacy (%) = [(mean value of infected untreated group (G b)mean value of all infected treated groups (either prophylactic or therapeutic)/mean value of infected untreated group] x100[39]. Histopathological examination: The terminal one cm of the ileum and the whole lung tissue were taken from each mouse and fixed in 10% neutral formalin. Ileum and lung tissues were embedded in paraffin, sectioned, mounted on glass slides, immersed in xylene, then dehydrated in graded alcohol solutions and stained by Hematoxylin and Eosin (HE)[40]. Stained sections were assessed regarding any pathological changes in the tissues, degree of inflammation, and severity of infection. Immunohistochemical staining of CD3 in lung tissu