Chickens in the positive control group (G2) infected with Eimeria tenella but not treated exhibited lower body weight gain compared to the other experimental groups, indicating impaired feed intake (FI) and feed conversion ratio (FCR) as reported by Mohammed et al. (2021). Avian coccidiosis, characterized by watery and bloody diarrhea, is a significant factor contributing to body weight loss and reduced growth performance due to its detrimental effects on feed absorption and digestion (Dkhil, 2013; Gres et al., 2003). Furthermore, Eimeria infection can lead to varying degrees of parasite-induced necrotic enteritis, which creates an environment favorable for the proliferation of Clostridium perfringens, a highly dangerous pathogen (Wade & Keyburn, 2015).
Several studies have investigated the use of natural plants and their extracts to inhibit and suppress the sporulation of Eimeria tenella oocysts (del Cacho et al., 2010; Molan et al., 2009; Yong et al., 2020; Zaman et al., 2012). In our current study, the extract of Aloe vera gel demonstrated successful reduction of sporulated oocysts and yielded better results than the vaccinated group. The decrease in mean oocysts per gram of feces (OPG) values achieved by Aloe vera gel extract was comparable to the best-performing group (G6) treated with amprolium, an effective anticoccidial drug. G6 exhibited the highest significant (p ≤ 0.05) reduction in mean OPG values compared to the positive control group (G5). Similar findings have been reported by Mwale et al. (2005) and Narsih & Wignyanto (2012). Aloe vera gel is rich in various biologically active components such as carbohydrates, steroids (Ni & Tizard, 2004), minerals, Acemannan, enzymes, Chromones, saccharides, and vitamins C, E, A, B12, B1, and B2 (Lawless & Allen, 2000). It also contains proteins such as lectins and lectin-like substances, as well as amino acids like alanine, arginine, aspartic acid, and glutamic acid (Choi & Chung, 2003), along with other miscellaneous organic compounds. Additionally, Aloe vera significantly enhances the growth performance of chickens by improving body weight (BW) and body weight gain (BWG) without inducing BW losses. Furthermore, it mitigates the adverse effects of vaccines on body performance, as vaccinated chickens often exhibit lower BW and BWG (Arczewska-Włosek & Świątkiewicz, 2014; Lehman et al., 2009).
The positive control group (G5) in our study, consisting of birds infected with Eimeria tenella, exhibited typical clinical signs associated with E. tenella challenge, including depression, weakness, pallor, bloody diarrhea, anorexia, and ruffled feathers. Additionally, there was a significantly high caecal lesion score, and the mortality rate reached 24% (Awais et al., 2012; Kim et al., 2013; Yong et al., 2020). Histopathological examination of the caecal lesions in G5 revealed necrosis, sloughing of epithelial cells, congested blood vessels, hemorrhage in the mucosa and submucosal layers, fibrous connective tissue proliferation, and edema. Numerous developmental stages of the parasite, such as oocysts, gametes, zygotes, schizonts, and free merozoites, were observed in large numbers, consistent with previous descriptions (Chanie et al., 2009; Ellakany et al., 2011). Notably, clusters of large schizonts were also present in the cecum, which are considered the second-generation schizonts responsible for extensive tissue damage, bleeding, disruption of caecal glands, and destruction of the mucosa and muscular layers (Ali et al., 2019).
Interestingly, chickens receiving Aloe vera as a prophylactic measure (G4) exhibited reduced caecal lesion scores, lower mortality (12%), decreased oocysts per gram (OPG) values, and mild to moderate diarrhea compared to the positive control group (G5). Histological examination of the cecal tissue in G4 revealed normal cecal villi, mild necrosis and degeneration of the submucosal glands, degenerated oocysts, and a decreased number of Eimeria stages in the mucosa and submucosa. These findings suggest that the growth and development of E. tenella were suppressed or negatively affected by Aloe vera. This may be attributed to the antiprotozoal activity of Aloe vera gel, which contains biologically important compounds and exhibits anti-inflammatory, anti-allergic, and antioxidant properties (Hu et al., 2003). Aloe vera also possesses antiseptic properties (Suga & Hirata, 1983), antibacterial effects (Alemdar & Agaoglu, 2009), as well as antiviral and antifungal properties (Davis, 1997), which aid in healing and protecting infected host tissues from injuries caused by Eimeria tenella and other pathogens.
Aloe vera exhibited a favorable effect against Eimeria tenella infection, demonstrating efficacy comparable to or superior to the anticoccidial drug amprolium alone (G6) or in combination with the anticoccidial vaccine Coccivac B (G2). Incorporation of Aloe vera gel as a prophylactic treatment in G4 or in combination with oocysts B in G3 resulted in improved caecal histopathological lesions, characterized by the destruction of many schizonts, disappearance of Eimeria stages, and a lower number of Eimeria oocysts.
Similar conclusions have been reached by Dardi (2018), McAllister et al. (1998), and Patterson and Burkholder (2003), who explained the synergistic effect of combining a coccidial vaccine with probiotics, prebiotics, and natural products such as Yucca schidigera extract. This combination helps maintain the integrity of intestinal villi, improve weight gain, and enhance feed conversion ratio.
Regarding red blood cell count and hemoglobin concentration, a significant difference was observed among the experimental groups after 9 days of infection. Aloe vera without infection resulted in the highest hemoglobin concentration and red blood cell count, even significantly higher than the non-infected non-treated control group. Conversely, the infected non-treated group (G5) exhibited the lowest values. The decrease in red blood cell count is attributed to severe bleeding and tissue damage in the intestinal mucosal surface during the acute stage of infection, as well as the release of large quantities of histamine, which increases capillary and venule permeability, leading to fluid exudation (Padmavathi and Muralidharan, 1986).
Among the experimentally infected and treated birds, group 6 (treated with amprolium) showed the highest hemoglobin concentration and red blood cell count, followed by Aloe vera, with no significant differences observed. These findings are in line with Haider & AL-Saegh (2018), who reported that hemoglobin concentration and red blood cell count significantly decreased in the infected non-treated group, while supplementation of Aloe vera juice in drinking water resulted in a significant increase in Hb and RBC values compared to the control groups.
The improvements observed in hematological and histological parameters can be attributed to the anticoccidial effects of Aloe vera and its content of biologically important compounds, including anti-inflammatory, anti-allergic, and antioxidant activities (Hu et al., 2003), as well as antiseptic properties (Suga & Hirata, 1983), antibacterial effects (Alemdar & Agaoglu, 2009), and antiviral and antifungal properties. Furthermore, Talmadge et al. (2004) stated that Aloe vera carbohydrates exhibit hematopoietic stimulating activities.
The infected groups in our study exhibited an increased total leukocyte count, which can be attributed to the direct effect of the infection, as infections generally lead to an increase in leukocyte count. However, more effective treatments resulted in a reduction in leukocyte count after 9 days of infection. These findings are in accordance with Melkamu et al. (2018), who reported a significant increase in total leukocyte count in broilers infected with E. tenella.
Conversely, Aloe vera treatment in the infected group led to a decrease in the total leukocyte count compared to the infected non-treated birds. This finding is consistent with the studies conducted by Mahdavi et al. (2012) and Yadav et al. (2017). The decrease in leukocyte count in the infected treated group suggests that the reduction in parasite load (due to the effect of Aloe vera) resulted in the down-regulation of immune system activity, leading to a decrease in the inflammatory response and subsequently a decrease in total leukocyte count.
In our study, chickens challenged with Eimeria tenella infection (G5) exhibited a significant up-regulation of IFN-γ, NO, and IL-4 levels at 9 days post-infection compared to the negative control group (G8). These cytokines are known to be directly involved in the immune response to E. tenella infection, and similar findings have been reported previously (Hong et al., 2006; Tian et al., 2014). The increased production of these cytokines in response to E. tenella infection is attributed to the up-regulation of their transcription levels (Hong et al., 2006; Yun et al., 2000). IFN-γ, produced by natural killer T cells, is believed to stimulate neutrophils and macrophages to migrate from circulation to the site of infection for the destruction of Eimeria sporozoites. Additionally, NO is involved in immunity and resistance against infectious diseases, as it exhibits toxicity towards certain parasites and bacteria (Allen & Fetterer, 2000). The significant down-regulation of these cytokine levels in G4 (infected Aloe vera treated) compared to the positive control group (G5) confirms the anti-inflammatory effect of Aloe vera extracts. The increase in serum NO level in G9 (unchallenged Aloe vera treated) compared to the negative control group (G8) further supports the direct role of Aloe vera in elevating NO levels in G4 (challenged and Aloe vera treated), leading to a reduction in inflammation (Allen & Fetterer, 2002).
Overall, the anticoccidial effect of Aloe vera in our study was comparable to or even superior to other herbal remedies that have been considered effective for coccidiosis control (Messaï et al., 2014; Yong et al., 2020; Zaman et al., 2012). Aloe vera supplementation significantly reduced fecal oocyst shedding by increasing NO levels and decreasing IL-4 and IFN-γ levels, which resulted in a reduction in inflammation and mitigation of the adverse effects of Eimeria challenge on growth performance, tissue injuries, and lesion scoring. Therefore, Aloe vera can be considered as an effective alternative therapy for avian coccidiosis control, as it successfully reduces sporulated oocyst shedding and mortality in chickens.