Cryptosporidiosis is among the most prevalent infectious diarrheal diseases in immunocompromised patients globally. In underdeveloped nations,
Cryptosporidium spp. ranks as the second predominant cause of infectious diarrheal illnesses among children under five years old
(Gebretsadik et al., 2018). Targeting and designing new nanoparticle dependent drug carriers achieves enhancement of treatment efficacy (Abd El
Wahab et al., 2021). In this study, the therapeutic effect of nanocomposites from cellulose nanocrystal-
Zingiber officinale extract supported by zinc oxide nanoparticles (Bio-ZnNPs) were evaluated against infected mice with
C.
parvum indicated by oocysts excretion, hematology, blood biochemical analysis, serum immunoglobulins levels and histopathological studies.
In this study, using the modified Ziehl Neelsen stain revealed that the stained
C.
parvum oocysts had a spherical to oval morphology, characterized by a pink hue on a blue-green backdrop (Fig 1). In the current study, the therapeutic use of Bio-ZnNPs against cryptosporidiosis revealed a lower significant (P<0.05) OPG count excretion from the first to the last day of treatment (1-5 DPT) in comparison to the Inf group. Bio-ZnNPs group had 47.2±0.25 x103 OPG, while NTZ group had 92.00±2.59x103 OPG (Fig 2).
Abouel-Nour et al., (2015) and
Abouelsoued et al., (2020) reported that oocyst shedding in ginger-treated mice gradually declined until it completely stopped. Also, Ginger and its phytochemicals were tested for its anti-cryptosporidiosis action
(Batiha et al., 2020). In addition,
Dkhil et al., (2015) discovered that ZnO-NPs decreased oocyst excretion and jejunal inflammatory damage in mice infected with
Eimeria papillata. This the same of
Mohi-Eldin et al., (2018), who said that
Allium sativum loaded ZnO-NPs decreased the oocyst in feces of rabbits infected with
Eimeria stiedae. Ginger medication may directly affect parasite growth in the intestines, sexual stage production and oocyst generation, reducing and eliminating fecal oocyst shedding
(Abdelgelil et al., 2023). The antibacterial efficacy of zinc oxide particles is believed to stem from the production of reactive oxygen species
(Sabir et al., 2014). Joe et al., (2017) shown that ZnO nanoparticles can compromise the oocyst wall glycoproteins, enabling penetration and hence elevating the release of detrimental Zn2+ ions.
Hematological studies revealed a substantial reduction (P<0.05) in RBCs count, Hb level and PCV percentage in mice infected with
C.
parvum oocysts against the control group on 5 DPT (Table 1). The Bio-ZnNPs treated group exhibited significant rise on 5 DPT in their levels in relation to the Inf group (Table 1).
Kabu (2023) demonstrated that hemoglobin levels were elevated during therapy, potentially linked to significant fluid loss. Despite the observation of alterations in hemoglobin, red blood cell counts and packed cell volume in calves suffering from diarrhea caused by Cryptosporidiosis, all hematological parameters remained within the established reference ranges (
Aiello, 2016). Besides, in this study, TLC and differential leukocytic count, comprising lymphocytes (%) and eosinophils (%), in the Inf group compared to the control group (Table 2). However, significant reductions were noted in the Bio-ZnNPs treated group, related to the Inf group on 5 DPT (Table 2). Nanoparticles initially engage with blood and its constituents, potentially inducing various immunogenic responses, inflammation and alterations in hematological parameters, including leukocytes
(Kim et al., 2009). The alterations in white and red blood cells documented herein may be attributable to an augmented immunogenic response
(Kawata et al., 2009) or disruptions in signaling pathways and cellular maturation
(Gopinath et al., 2010), which can influence red blood cells alongside the proliferation and differentiation of other cells.
Cryptosporidiosis in mice may result in a chronic disease state affecting the gastrointestinal system and/or extraintestinal locations, potentially causing hepatic impairment
(Mead et al., 1994). This study indicated that the Inf group had substantial elevations (p<0.05) in liver enzymes such as ALT, AST and ALP compared to the control group. In contrast, the Bio-ZnNPs group showed a significant (P < 0.05) drop in the three enzyme levels relative to the Inf and NTZ groups on 5 DPT (Fig 3). These findings corroborated that cryptosporidiosis may have extraintestinal consequences (
Chalmers and Davies, 2010).
Malekizadeh et al., (2012) discovered that a 3% supplementation of ginger powder reduced serum ALT and AST activity in laying hens. Similar benefits of ginger were noted in broilers
(Sahoo et al., 2019) and laying quails
(Herve et al., 2019). The enhanced hepatic function may be ascribed to the antioxidant chemicals, including 6-gingerol, present in ZOE. Prior research shown the preventive properties of 6-gingerol against oxidative stress-induced liver damage both in vitro and in animal
(Joshi et al., 2017; Vipin et al., 2017). The radical-scavenging activity of antioxidant compounds in ZOE may elucidate this phenomenon, as they inhibit lipid peroxidation
(Si et al., 2018) and enhance organ function, as indicated by reduced ALT and AST serum activities, thereby promoting the synthesis of antioxidant enzymes
(Li et al., 2019). Mahmoud et al., (2021) observed no adverse effects on liver histology, blood physiology, immunological function, or DNA integrity in broilers following prolonged low-dose ZnO NP administration. Zinc treatment restored AST and ALT activity, demonstrating its hepatoprotective impact by controlling protein synthesis. Zinc’s antioxidant capabilities can also protect hepatocytes from adverse effects and reduce enzyme leakage into the bloodstream
(Kouadria et al., 2020).
In the
C.
parvum infection group, sera levels of IgG and IgM increased with a significant (p<0.05) difference in comparison to the control group. On the other hand, the Bio-ZnNPs group showed the lowest blood titres of IgG and IgM than NTZ group, compared to the Inf group (Fig 4).
Khan et al., (2004) indicated that diarrhea was associated with lower IgA and IgM levels. Ginger therapy modulated immunological responses, according to
Abdelgelil et al., (2023). Piglets’ blood IgM and IgG levels improved with ZnO and nano ZnO supplementation
(Sun et al., 2019). Zinc strongly affects humoral and innate immune activity (
Knoell and Liu, 2010). Serum IgG and IgM can protect the extravascular compartment against pathogenic viruses and bacteria
(Li et al., 2007). Nanoparticles has the ability to treat experimental cryptosporidiosis through disruption of the normal physiological bioactivities of the parasite, such as food absorption, reproduction and mobility
(Hamdy et al., 2023). Immune system enhancement by stimulating the natural killer cells’ activity and phagocytosis may be another mechanism (
Gaafar, 2012).
Furthermore, in the present study, histopathological examination results showed that the control group exhibited normal ileal tissue architecture, characterized by normal villi and goblet cells (Fig 5A). additionally, the Inf group with
C.
parvum displayed pronounced villous atrophy and sloughing epithelium, characterized by a considerable presence of goblet cells, alongside severe intraluminal infection by
Cryptosporidium oocysts and significant infiltration of inflammatory cells (Fig 5B). Similar results were reported by
Bhagat et al., (2017) and
Sood et al., (2019). Conversely, the Bio-ZnNPs treated group displayed almost normal elongated ileal villi, well-organized intestinal crypts and a reduced quantity of goblet cells, with an absence of intraluminal
Cryptosporidium oocysts (Fig 5C). On the other hand, the NTZ treated group exhibited regenerative moderate crypts generating new villi, with the presence of goblet cells (Fig 5D). Various plant extracts, including garlic, ginger, curcumin and black seeds, were evaluated on Cryptosporidium-infected experimental mice and shown an anti-Cryptosporidial effect by safeguarding the intestinal epithelial tissue
(Abouel-Nour et al., 2015; Asadpour et al., 2018;
Sadek et al., 2020). Plant extracts may inhibit or compete for receptor sites on the gut surface, hence reducing
C.
parvum colonization
(Harp et al., 1996). The pronounced positive and rectified impact of ginger in the current investigation may be due to its anti-inflammatory activities, notable antioxidant and immunomodulatory capabilities
(Adewusi et al., 1996). Ali et al., (2008) elucidated the immunological effects of ginger, ascribed to its active constituents, including zingerone, paradol, gingerols and shogaols. The most promising biopolymers in nanotechnology are natural substances like CNCs. This nanomembrane material is optimal owing to its superior mechanical strength and toughness
(Li et al., 2021). CNCs are biopolymers that ensure safety for medical applications such as medication delivery systems
(El-Shafai et al., 2022; Mehany et al., 2022). Sharaf-El-Deen et al., (2023) indicated that ZnONPs inhibited the attachment of
C.
parvum oocysts to the intestinal tissue of mice.
Hamdy et al., (2023) noted that animals treated with
A.
sativum-loaded ZnO-NPs demonstrated the most pronounced enhancement in histological intestinal lesions with no
Cryptosporidium oocysts detected in the intestinal villi.
