Therapeutic efficacy of geranium-thymol combination against murine eimeriosis with reference to its apoptotic activity

1 Introduction

The members of the genus Eimeria are the most common cause of coccidiosis. This disease is one of the most prevalent parasitic diseases in poultry industry, causing large yearly losses of up to USD 13 billion per year (Blake et al., 2020). Eimeria is an obligatory parasite in the phylum Apicomplexa with exogenous and endogenous life cycle phases. These parasites can infect and proliferate inside the mucosal epithelia in various parts of the gut via the oral route. As a result, they induce gastrointestinal injury (i.e., inflammation, watery/bloody faeces, etc.), morbidity, and mortality in poultry (Chapman, 2014). This pathogen can disturb the intestinal microbiota and predispose the intestinal environment to the growth of pathogenic bacteria such as Clostridium perfringens, resulting in necrotic enteritis (Chapman, 2014).

The primary strategies of preventing and controlling this infection include anticoccidial drugs such as ionophores, synthetic chemicals, coccidiocides, and vaccination(Chapman et al., 2010). Resistance has been established for all of the medications now in use, thus the development of innovative agents with distinct mechanisms of action is critical if chemotherapy is to remain the primary way of controlling this disease (Muthamilselvan et al., 2016; Thagfan et al., 2023). Furthermore, the use of anticoccidial drugs as feed additives for prevention has been rigorously prohibited in European countries since 2006, with a complete prohibition expected to take effect in 2021 (European Council Council Decision 2011/50/EU).

Essential oils (EOs) or aromatic compounds could be appropriate alternative strategies to control coccidiosis (Namdeo et al., 2020). Thymol (T) is a naturally occurring phenol monoterpene. It is one of the most significant nutritional components of thyme species (Nagoor Meeran et al., 2017). It has a long history of usage in traditional medicine and has been demonstrated to have a broad spectrum of pharmacological effects, including the treatment of neurological and respiratory disorders (Gavliakova et al., 2013, Yu et al., 2016,), antiparasitic and antihelminthic (Tisserand and Young, 2014), antioxidant, anticancer (Hashemipour et al., 2013).

Pelargonium graveolens (PG) is a medicinal herb in the Geraniaceae family. This plant's essential oil has a wide range of biological benefits, including insecticidal, antibacterial, antifungal, and antiparasitic characteristics. It is also used in aromatherapy to treat gastrointestinal disorders (Huang et al., 2021).

According to Remmal et al. (2013) and Boyko et al. (2021), both PG and T have oocysticidal activity against chicken Eimeria. As a result, the current study is intended to test the potential efficacy of a geranium-thymol mixture in the management of murine eimeriosis caused by Eimeria species of mice through in-vitro and in-vivo trials.

2 Material and methods

3 Result

3.2

3.2 Effect of PGT on faecal oocyst shedding

On day 5p.i., faeces oocyst output peaked at around 6761.66 × 10³ ± 372.34 oocysts/g faeces in the infected group. In the groups treated with 50, 100, and 150 mg/kg of PGT suspension, oocyst output was reduced by 4.4, 23.59, and 56.92 %, respectively (Fig. 1). The 150 mg/kg dose was clearly the most effective in reducing faeces oocyst discharge. As a result, we only used the 150 mg/kg dose in subsequent studies.

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Fig. 1

Effect of PGT at varying doses (50, 100, and 150 mg/kg) on the oocyst output patterns of mice infected with E. papillata on day 5p.i. (Data are given as Mean ± SEM). Significant difference as compared to control (P ≤ 0.05).

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3.3

3.3 Histological observations

Treatment with 150 mg/kg PGT resulted in a 66.19 % (P ≤ 0.001) reduction in the number of parasite stages (meronts, microgametes and macrogametes) per ten villous-crypt units and 56.9 reduction in the number of shedded oocysts when compared to the infected group (Figs. 2-4), (Table 2).

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Fig. 2

Effect of PGT at 150 mg/kg on the numbers of parasitic stages (A) and amount of shedded oocysts (B). (Data are given as Mean ± SEM). Significant difference as compared to control (P ≤ 0.05).

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Fig. 3

Histological sections of mouse jejunum stained with hematoxylin and eosin showed the effect of PGT at 150mg/kg on the parasitic stages of E. papillata.  (A) Uninfected control group; (B) Infected group showed high numbers of developmental stages; (C) Infected treated group with reduced numbers of developmental stages. Scale-bar = 50 µm.

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Fig. 4

Section of mouse jejunum infected with E. papillata and, stained with hematoxylin and eosin on day 5p.i showing different developmental stages in inner epithelium. meronts (Me), macrogamont (Ma), microgamont (Mi) and developing oocyst (Do). Scale-bar = 100 µm.

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Table 2 Parasitic stages in jejunum of E. papillata-infected and infected-treated mice with PGT.

Group	Meronts / 10 VCU	Male and female gamonts/ 10 VCU	Developing Oocysts / 10 VCU
Infected	78.00 ± 3.74	46.00 ± 4.00	18.00 ± 2.00
PGT	33.00 ± 4.36***	11.00 ± 1.55***	4.00 ± 1.18***

All values are means ± SEM. *** significance (P ≤ 0.001) between the infected and treated groups.

3.4

3.4 Apoptosis and apoptotic score

PGT treatment significantly reduced the number of apoptotic cells in the jejunum tissue (Fig. 5.), from 53.33 ± 8.81 to 20.00 ± 5.77 apoptotic cell/10 VCU (P ≤ 0.05) (Fig. 6).

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Fig. 5

Effect of PGT on the apoptosis level in jejunum of mice. Apoptotic cells in control (A), E. papillata infected (B), and infected-treated (C). Black arrows indicating TUNEL positive cells. Scale-bar = 50 µm.

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Fig. 6

The number of apoptotic cells in different group. Significant change at P ≤ 0.05 with respect to the non-infected and the infected group.

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3.5

3.5 Effect of PGT on oxidative stress in jejunal tissue

The infection of mice with E. papillata significant decrease in the glutathione (GSH) levels (P ≤ 0.001). When compared to infected groups, PGT treatment resulted in a significant increase in GSH (P ≤ 0.001) (Fig. 7A). Also, the infection caused a significant increase (P ≤ 0.01) in MDA levels while the treatment with PGT improved these MDA alterations (P ≤ 0.05) (Fig. 7B).

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Fig 7

Effect of PGT on the level of reduced glutathione (A) and malondialdehyde (B) in mouse jejunum infected with E. papillata. (Data are given as Mean ± SEM). Significant difference as compared to control and infected group (P ≤ 0.05).

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3.6

3.6 Sporulation inhibition test

The microscopic examination and counting of oocysts treated with PGT at various concentrations resulted in deformed oocysts with cracked walls and lysis (Fig. 8). The sporulation rate for the control after three days was 83 % ± 1.73. At the doses of 2.5 and 1.25 % (P ≤ 0.05), PGT significantly reduced sporulation rate (P ≤ 0.05) and it totally stopped at a dose of 5 %. The sporulation inhibition rates of PGT relative to control were 100 %, 81.13 %, and 61.85 % for 5, 2.5, and 1.25 mg doses, respectively (Table 3). Also, the oocysticidal effect was confirmed by the higher ratio of degenerated oocysts 96.33, 46.00 and 16.66 for 5, 2.5 and 1.25 % of PGT doses, respectively compared to the control. The liner equation showed the highest coefficient of determination (R²) values (P ≤ 0.001) for sporulation and destruction (Fig. 9).

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Fig. 8

The morphology of E. papillata oocysts treated with PGT different concentrations. Typical sporulated oocyst from control (A). Destructed oocysts after incubation with PGT 5 % (B, C). Abnormal oocysts after incubation with PGT 2.5 % (D, E, F). Degenerated oocysts after incubation with PGT 1.25 % (G, H). Scale-bar = 10 µm.

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Table 3 In-vitro effect of PGT different concentrations on sporulation rates of E. papillata oocysts.

Concentration/ Group	% Sporulated oocysts	%Unsporulated Oocyst	% Destructed oocyst
control	83.00 ± 1.73a	15.66 ± 1.45c	1.33 ± 0.33d
PGT 5 %	0.00 ± 0.00d	3.66 ± 0.88c	96.33 ± 0.88a
2.5 %	15.66 ± 2.3c	41.66 ± 7.26b	46.00 ± 6.65b
1.25 %	31.66 ± 1.66b	55.00 ± 2.88a	16.66 ± 1.66c

Means with different superscripts within a column are significantly different at P ≤ 0.05.

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Fig. 9

Liner correlation between sporulated and concentration of PGT oil (A), and destructed oocyst and concentration of PGT oil (B).

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4 Discussion

The use of EOs and their metabolites may be an alternative tool to anticoccidial drugs for coccidiosis management (Githiori et al., 2006). Furthermore, the synergistic effects of the various compounds in EOs are another important aspect that demonstrated a greater mode of action than individual compounds. Because the dose of use is reduced, the synergism serves to lessen the environmental and toxicological impact (Haleem et al., 2019).

According to the GC–MS analysis of PG, its main components were citronellol (14.44 %), geraniol (11.08 %), and linalool (7.74 %). Citronellol and geraniol are the key components responsible for PG's antiparasitic action (de Mello et al., 2023).

In this study, the synergism between P. graveolens essential oils and thymol against E. papillata infection was investigated. Different doses of PGT (50,100,150 mg/kg) have been tested. In terms of anticoccidial activity, PGT might impede E. papillata development in the host and finally oocyst excretion in mouse faeces, with the highest dose (150 mg/kg) being the most potent.

According to the previously discussed issues, EOs have a high oocysticidal activity due to their hydrophobic characteristics and low molecular weight, as well as their bioactive compounds, which can destroy parasites including oocysts and sporozoites (Abbas et al., 2012, Quiroz-Castañeda and Dantán-González, 2015, Muthamilselvan et al., 2016).

Thymol showed oocysticidal action against Eimeria species of pigeons, with oocyst counts significantly decreased at 10 % thymol concentration. While P. graveolens did not prevent E. magna sporulation, 55–75 % of E. magna oocysts sporulated after being exposed to P. graveolens essential oils (Remmal et al., 2011, Remmal et al., 2013, Arafa et al., 2020). Also, Sárközi et al. (2007) found that thymol has antiparasitic activity against Eimeria spp., and that their mode of action is linked to the degradation of the sporozoite membrane and the subsequent loss of calcium ions from the parasite, which is essential for invasion. Furthermore, Küçükyilmaz et al. (2012) discovered that oregano essential oils, which are high in thymol and carvacrol, help to improve animal health during coccidia challenge and lower the quantity of oocysts shed in faeces. Similarly, thymol may inhibit enzymes involved in protozoal metabolism, such as dihydrofolate reductase. This enzyme catalyses the reduction of dihydrofolate to tetrahydrofolate, which is a precursor of cofactors needed for the biosynthesis of purines, deoxythymidine triphosphate (dTTP), and a number of amino acids in a NADPH-dependent manner. As a result, inhibiting dihydrofolate reductase causes cell growth to stop and death to occur (Hikal et al., 2021).

The histological analysis conducted in this study indicated that E. papillata infection damaged the intestinal tissue at the infection site. This damage was caused by the parasite's developmental stages, particularly merozoites, which broke out of the intestinal cells to invade other intestinal cells (Al-Quraishy et al., 2019).

Intestinal eimeriosis causes oxidative damage and antioxidant depletion, which leads to irreversible cell damage due to an increase in reactive oxygen species, which is associated with an increase in lipid peroxidation, protein oxidation, damaged DNA and cellular membranes, and ultimately cell death (Georgieva et al., 2006, El-Shahat et al., 2009). Essential oils can indirectly interfere with parasitic metabolism by increasing the host immune response and antioxidant defense systems, allowing for effective parasitic invasion control and eradication (Idris et al., 2017).

Our findings showed that PGT reduced oxidative stress in the infected jejunum, which was supported by its antioxidant activities. Similarly, Abdel Rahman et al., 2020 discovered that thymol and geranium supplementation enhanced antioxidant status and reduced MDA levels in broilers and common carp. Apoptosis is critical in the gastro intestinal system because it allows for the continual and rapid replacement of intestinal cells while maintaining tissue homeostasis. Nevertheless, several variables, including increased reactive oxygen and nitrogen intermediates and inflammatory cytokines within cells, contribute to apoptosis (Major et al., 2011). Our results indicated that the number of apoptotic cells in the jejunum of infected mice treated with PGT is significantly lower than in infected mice. Similarly, Green, 2000 demonstrated that E. papillata causes apoptosis, which might be attributed to the complicated host-parasitic relationship; parasite invasion and multiplication, which could cause significant stress to the host cell (Metwaly et al., 2014). PGT have substantial antioxidant and anti-inflammatory properties and thus reduce E. papillata-induced apoptotic effects in infected mice jejuna (El Aanachi et al., 2020, Gholami-Ahangaran et al., 2022).

The in-vitro study, our findings revealed the presence of destructed and deformed oocysts with cracked walls in the oocysts treated with PGT. Also, the rate of oocyst sporulation was significantly lower than in the control group, particularly at 5 % PGT dosage. These findings suggested that PGT could destroy a cell's permeable outer membrane (Boyom et al., 2003). The increased cytoplasmic membrane permeability promotes the release of lipopolysaccharides, porin proteins, cellular fatty acids, and the leaking of metabolites and enzymes such as aromatic amino acids and nucleotides (Remmal et al., 2011). As a result of their hydrophobic abilities, oocysts degenerate as a result of greater cell membrane penetration (Boyom et al., 2003). Therefore, essential oils have anticoccidial activities by diffusing across parasite cell membranes, causing permeability disruption of cell membranes, and interfering with cell metabolisms. The process causes ion, energy, and other cell components to seep out, resulting in cell death (Allen et al., 1997), and also induction of oxidative stress, which inhibits invasion and impairs Eimeria growth (Abbas et al., 2012). In addition, geraniol and citronellol the main component of geranium oil can act on cell wall and cell membrane via interfering with ergosterol biosynthesis (Pereira et al., 2015).

In conclusion, mixing geranium oil and thymol oil may be an effective way to boost the anticoccidial effectiveness of natural extracts. Our findings suggest that PGT decreases developmental stages, oocyst output, and can impair E. papillata sporulation. This protective impact could be attributed to PGT's antioxidant and anti-apoptotic characteristics.

CRediT authorship contribution statement

Heba AbdelTawab: Writing – original draft. Shawky M. Aboelhadid: Supervision, Writing – original draft. Almahy M. ElMallah: . Abdel-Azeem S. Abdel-Baki: Supervision, Writing – original draft. Saleh Al-Quraishy: . Ahmed O. Hassan: . Ebtesam A. Yousef: .