Moringa oleifera, Ocimum gratissimum and Vernonia amygdalina as a natural antiparasitic alternative in growing rabbits

Pascal Abiodoun Olounladé¹, Basile Boni Saka Konmy^1,2, Erick Virgile Bertrand Azando³, Sanni-yô Doko Allou¹ and Lamine Baba-Moussa²

¹ Zootechnical Research and Livestock System Unit, Laboratory of Animal and Fisheries Science (LaSAH), National University of Agriculture, 01 BP: 55 Porto-Novo, Benin
abiodouno@yahoo.fr
² Laboratory of Biology and Molecular Typing in Microbiology, Faculty of Science and Technology, University of Abomey-Calavi, UAC, 05 PO Box 1604, Cotonou, Benin
³ Laboratoire d’Écologie, de Santé et de Productions Animales, Département des Sciences et Techniques de Production Animale et Halieutique, Faculté d’Agronomie, Université de Parakou, 01 BP 2115, Cotonou, Bénin

Abstract

Coccidiosis is known as one of the parasitic diseases that inflict economic loss on rabbits’ farms. Coccidiosis is controlled with chemical agent. Due to the excessive use of anticoccidials, drug resistance has been developed. This study aims to assess the activity of Moringa oleifera, Ocimum gratissimum and Vernonia amygdalina on the elimination of coccidia cysts and helminth eggs in rabbits. 40 New Zealand rabbits, 60 days old and 1100 ± 122g body weight and naturally infected were used. The rabbits were assigned to forth groups of ten rabbits per group. The first three groups: Mo, Og and Va received respectively Moringa oleifera, Ocimum gratissimum and Vernonia amygdalina leaves and stalks ad libitum for three days and the fourth group (control group) received no plants. The McMaster technique was used to count helminth eggs and oocysts. Data were analyzed using MANOVA and ANOVA methods in SPSS 26 Software. This study demonstrates that oral administration of those plants leaves and young stalks reduced the release of oocysts and eggs per gram of faeces. This significant effect (p<0.001) was observed at day 1 post treatment for three plants. The efficacy of both plants Moringa oleifera and Vernonia amygdalina was 100% for helminths and coccidia. However, the efficacy of Ocimum gratissimum was 99.85% for coccidia and 97.92% for helminths. The results indicate those leaves and young stalks plants could be a natural alternative for the coccidiosis and intestinal worms’ control in rabbit production.

Key-words: plants, antiparasitic, growing rabbits, Benin

Introduction

Rabbit production is a fast growing activity in Benin (FAO 2018b) especially amongst smallholder breeders (FAO 2018a). Gastrointestinal parasitosis remain one of the most common rabbit production constraints in many developing countries (Kpodekon et al 2015). Currently for treatment of coccidiosis, sulfonamides, salinomycin and robenidine are mainly used (Pakandl 2009), which may become toxic to infants and pregnant females. The problem with commercial and synthetic anticoccidial is not confined to anticoccidial resistance alone. The indiscriminate application of such anticoccidial also threatens the public health by the increasing reports of drug residues in meat. Moreover, the expensive product poses a question about its sustainability in developing countries where rabbits breeding is common (Adediran et al 2014; Abbas et al 2017; Arlette et al 2019). Nowadays more importance is given to the use of herbs and spices in animal nutrition, as possible replacement of antibiotics, the latter – as growth promoters – banned in the European Union since 2006. These natural products supplemented to livestock diets have shown to stimulate a wide range of favourable effects, such as digestive stimulation, anti-microbial, anti-viral, growth promoter and antioxidant (Dalle Zotte et al 2016).Moringa oleifera, Ocimum gratissimum and Vernonia amygdalina were one of the important plants in the Benin utilized for food and traditional medicine (Cabardo and Portugaliza 2017; Monga et al 2017; Adefisayo et al 2018).

The anticoccidial activity study of Vernonia amygdalina fresh leaves aqueous extract did reveal a Eimeria tenella oocyst reduction potential in chicken (Dakpogan et al 2019). The leaves and flower of Ocimum gratissimum are traditionally used as digestive, stomachic and tonic agents. Ocimum gratissimum have been recommended as antimicrobial, antifungal, antibacterial, antimalarial, antiviral, anesthetic, antiprotozoal and anthelmintic agents (Monga et al 2017). de Lima Boijink et al (2016) reported that the essential oil of Ocimum gratissimum is a safe and efficient ingredient to be used in natural anthelmintic and anesthetic products for tropical fish farming. However, the essential oil of Ocimum gratissimum, were efficient in inhibiting hatching of H. contortus eggs, showing possible utilizations in the treatment of gastrointestinal helminthosis of small ruminants (Pessoa et al 2002).

The aim of the present study was to assess the activity ofMoringa oleifera, Ocimum gratissimum and Vernonia amygdalina on the elimination of coccidia cysts and helminth eggs in rabbits.

Material and methods

Plant collection

Leaves and stalks of Moringa oleifera, Ocimum gratissimum and Vernonia amygdalina were collected in Faculty of Agronomics Sciences farm, University of Abomey Calavi and identified at National Herbarium of Benin using a voucher specimen registered under the reference N° AA66/1645/HNB; YH524/HNB and YH523/HNB respectively. The leaves and stalks of those plants were harvested and served fresh to the animals.

Experimental Animals

40 New Zealand white rabbits approximately 60 days of age with 1100 ± 122gr of body weight were used, and the rabbits were naturally infected and belonged to a farm with a history of intestinal coccidiosis. Rabbits were kept in individual galvanized cages of size 80 cm wide × 50 cm long × 40 cm high and fed with a commercial feed. The handling of animals was according to international bioethical standards. Before the beginning the experiment, the Eimeria species were identified using a flotation technique and the oocysts per gram of feces (OPG) were quantified with the technique of McMaster in order to confirm the natural infection by helminth eggs and larvae and coccidian cysts (Eimeria spp). After two weeks, the animals were randomly assigned to three group treatments (Mo, Og and Va) consisting of ten rabbits each and a control group (Ct) of ten rabbits too.

The rabbits in the treatment groups Mo, Og and Va were fed withMoringa oleifera, Ocimum gratissimum and Vernonia amygdalina leaves and stalk ad libitum respectively for three days while the control group was denied access to the plant. Faecal samples from all the rabbits were collected after days 1, 2, 4, 7, 14 and 21. The samples were then sent to the laboratory for analysis.

Evaluation of Anticoccidial Activity

Fecal samples were collected with gauze placed under the cages and then placed in polyethylene bags and transferred to the laboratory at 4°C. This methodology was performed at 0, 2, 5, 7, 10, 13, 16, 19 and 22 days of experimentation. A morphological and morphometric identification of Eimeria species present in the experimental groups was made using the flotation technique (Karamon et al 2008). The oocyst count, helminth eggs per gram of the feces (OPG and EPG) and helminth larvae were recorded and quantified according to McMaster techniques with four repetitions per group (Bortoluzzi et al 2018).

The percentage of antiparasitic efficacy was calculated using the formula modified from that described by (Arundel et al 1985) as follows:

Where N = Mean number of helminth egg or oocyst in control (untreated) animals.

n = Mean number of helminth egg or oocyst in treated animals.

Data management and statistical analysis

Data collected were stored in Microsoft Excel spreadsheet 2019. Were analyzed in a completely randomized design using a single factor univariate analysis of variance (ANOVA) with four levels of extract for each time. Multivariate analysis of variance for repeated measures was performed. Oocyst and eggs counts were natural log transformed, prior to analysis. The p < 0.05 was considered to be statistically significant.

Results

In this study, no adverse effects were physically observed throughout the treatment period in rabbits. However, two rabbits belonging to the control group died. The autopsy revealed the presence of intestinal lesions probably due to advanced coccidiosis.

Oocyst quantification

Coccidia oocysts ranged from 1084.5 ± 423.92 to 1725.5 ± 549.96 before treatment in both the treatment and control groups (Table 1). The number of coccidia oocysts had significantly decreased two days after treatment with plant leaves and stems. At the second day of treatment the OPG was 0.33 ± 0.52; 20.83 ± 7.55 and 0.67 ± 0.82 in the Moringa oleifera, Ocimum gratissimum and Vernonia amygdalina treated subjects, respectively. At this day (day 2), statistically differences (p<0,001) between the Control group with the groups receiving the leaves and stalk of those plants were found. At Day 4 the OPG recorded was 0 in the Moringa oleifera and Vernonia amygdalina groups and 2.5 ± 2.59 in the Ocimum gratissimum group. On day 7 all OPGs of the treated subjects were zero. However, in the control group an increase in the number of oocysts was recorded (Table 1). At day 21, statistically significant differences (P<0.05) among the three groups of plants were found, with lowers values with the group receiving Ocimum gratissimum leaves and stalks (Table 1).

Dunnet test was used for multiple comparisons (Figure 1). At day 2, statistically differences (P<0.001) in the log of average number of oocysts

Figure 1. Oocysts output in faeces of growing rabbits according to the groups and time (n=10 rabbits per treatment). A significant (P<0.001) difference was observed among groups from day 2 until the end of the experiment. OPG = Oocysts per gram in faeces

Egg counts

Helminth eggs observed during examination of droppings by the flotation and sedimentation technique were Graphidium strigosum, Trichostrongylus retortaeformis, Passalurus ambiguous, Cittotaenia Pectinata, C. denticulata and Coenurus pisiformis.

The analysis of variance showed a significant interaction between time and treatment (P<0.001) which means that the log average number of eggs per gram of faeces (EPG) for each treatment are not homogeneous behavior over time (Figure 2)

Figure 2. Eggs output per gram of faeces of growing rabbits according to the groups and time (n=10 rabbits per treatment). A significant (P<0.001) difference was observed among groups from day 1 until the end of the experiment (day 21). Helminths, number of Eggs per gram of faeces (EPG)

The egg per gram (EPG) values of both treated and control groups at the beginning of the experiment ranged from 412.50 ± 243.80 to 471.67 ± 128.98 (Table 2). After two days of treatment days rabbits given Moringa oleifera and Vernonia amygdalina leaves recorded 0 EPG while those given Ocimum gratissimum recorded 9 ± 5.97 EPG (Table 2).

At day 1, decreased (P<0.001) numbers of oocysts were obtained with groups receiving Moringa oleifera, Ocimum gratissimum and Vernonia amygdalina (Table 2). All treated groups recorded 0 EPG after three days of administration while EPG in the control group increased (Table 2). All rabbits developed helminthic infection, and the helminths observed in order of abundance were Graphidium strigosum (67%), Trichostrongylus retortaeformis (43%), Passalurus ambiguous (26%), Cittotaenia Pectinata (11%) , C. denticulate (4%) and Coenurus pisiformis (1,9%). From the first week of administration, all rabbits were free of helminthic infection and remained free throughout the one-month trial period.

The percentage of anticoccidial efficacy as calculated from the formula above. Each plant tested (Moringa oleifera, Ocimum gratissimum and Vernonia amygdalina) showed increasing efficacy as a function of time (days) during treatment against coccidial oocysts. The efficacy of the plants against coccidia oocysts was 99.85% for Ocimum gratissimum and 100% for Moringa oleifera and Vernonia amygdalina at day 4 post treatment (Figure 3a). The calculated efficacy against helminth eggs was 100% for Moringa oleifera and Vernonia amygdalina and 97.92% for Ocimum gratissimum at day 2 post-treatment (Figure 3b).

Figure 3. Efficacy of treatment of Moringa oleifera,Ocimum gratissimum and Vernonia amygdalina against coccidian cyst and helminths eggs. a: Efficacy on coccidian cyst; b: efficacy on helminths eggs

Discussion

The parasitic disease was a serious health and economic problem in rabbits in the world, affecting mainly young rabbits after weaning (Papeschi et al 2013). The use of natural antiparasitic like medicinal plants was a promising way to improve health and commercial rabbit production. The plants Moringa oleifera, Ocimum gratissimum and Vernonia amygdalina, were popular ethnomedicines with clinical significance that were widely used in certain countries and continents. Moringa oleifera, Ocimum gratissimum and Vernonia amygdalina were commonly used in tropical Africa. These plants function as anthelminthics and used to treat gastrointestinal disorders (Ademola and Eloff 2011; Kandil et al 2018; Aderibigbe and Idowu 2020; IfedibaluChukwu et al 2020). The present study used leaves and stalk plant, rather than single components isolated from the plant, because breeders generally take whole plant or aerial part to animals, usually containing several constituents. Furthermore, the leaves often contain components pharmacologically active components.

In the current work, the efficacy of Moringa oleifera, Ocimum gratissimum and Vernonia amygdalina in the treatment of helminths and coccidial infection in rabbits was evaluated. Previous studies demonstrated that medicinal of plants can suppress helminthiasis Adediran et al (2014) and coccidiosis by intervention with the developmental stages of life cycle in Eimeria species (Muthamilselvan et al 2016). The result obtained showed that Moringa oleifera, Ocimum gratissimum and Vernonia amygdalina has antihelminthic and anticoccidal effects in rabbits.

Studies on the anthelmintic effects of those three plants have been conducted by other researchers with different results. Kandil et al (2018) and Cabardo Portugaliza (2017) reported about 100% activity in rabbits and 95.89% activity in goat of Moringa oleifera respectively. Rastogi et al (2009) showed that anthelmintic activity against Indian earthworm ofMoringa oleifera was dose dependent. Adediran et al (2014) and Adediran Uwalaka (2015) reported about 100% activity of Vernonia amygdalina in goat. Alawa et al (2010) reported about 59% activity of this plant in cattle. Vernonia amygdalina may be used in alternative medicine for treatment of helminth-related diseases (Oyeyemi et al 2018). The plant provides an alternative and natural antiparasitic agent for goat helminth and coccidian that was environment friendly and without the effect of chemical residues (Adediran et al 2014). The anthelmintic effect in chicks of Ocimum gratissimum was dose dependent and was about 55.8% (Njoku and Asuzu 1998). de Lima Boijink et al (2016) showed that essential oil of Ocimum gratissimum was a safe and efficient ingredient to be used in natural anthelmintic and anesthetic products for tropical fish farming.

The efficacy of Moringa oleifera as an anthelminthic in rabbit (100%) was similar to the one reported by (Kandil et al 2018) in rabbit. However, this efficacy was higher than that reported 95% activity in goat (Cabardo and Portugaliza 2017). Vernonia amygdalina efficacy of this study (100%) was similar that reported by (Adediran et al 2014; Adediran and Uwalaka 2015) in goat. The result of efficacy Ocimum gratissimum effect as an anthelmintic (99.85%) was higher compared to 55.8% reported in chicken (Njoku and Asuzu 1998). Njoku Asuzu (1998)administered aqueous extract of the plant to the chicken while in this experiment rabbits were fed whole leaves and young stalks of the plant. This could probably account for the higher efficacy reported in this experiment as compared to that of (Njoku and Asuzu 1998). It could also be due to other factors that need to be considered such as genetic and physiological conditions.

Various reports on the effects of those plants as an anticoccidial agent in other species exists even in Nigeria, Cameroon, Egypt (Adediran et al 2014; Arlette et al 2019; Abu El Ezz et al 2020) but there has been no report on the coccidial species in rabbit. This study reported an efficacy of 99.85% for Ocimum gratissimum and 100% for both Moringa oleifera and Vernonia amygdalina as an anticoccidial agent in rabbit. This was higher than that reported by (Abdel-Tawab et al 2020) who reported efficacies of 57% of Astragalus membranaceus against Eimeria papillata infection in mice. Abu El Ezz et al (2020)reported that the efficacy of Moringa oleifera oil against hepatic coccidiosis was 55%. The author revealed that the highest titer of antibodies was detected in moringa oil treated rabbits. It was concluded that moringa oils had an anti-coccidial effect. Similar efficacy was obtained by (Adediran et al 2014) who reported efficacy of 99.4% of Vernonia amygdalina against Eimeria in goat. The plant provides an alternative and natural antiparasitic agent for goat helminth and coccidian that was environment friendly and without the effect of chemical residues. On the contrary, (Al-Fifi 2007) reported that the powder of Vernonia amygdalina leaves reduced the OPG to only 35%. These authors proposed that the anticoccidial effect of these plants could be attributed to its antioxidant properties and that the antioxidants constituents were flavonoids and vernosides. The results of this study were close to Rivero-Perez et al (2019) which revealed that Salix babylonica hydroalcoholic extract decreased the release of OPG in rabbits that were naturally infected with the Eimeria spp. This activity was due to its content of phytochemicals with anticoccidial properties such as coumarins, triterpenes, flavonoids, sesquiterpene lactone, saponins, terpinene, linalol, thymol, and carvacrol.

Conclusion

Moringa oleifera, Ocimum gratissimum and Vernonia amygdalina leaves and stalks added in feed of rabbits for three consecutive days decreased the release of OPG and EPG in rabbits that were naturally infected with the parasites. The best results on the reduction of OPG and EPG were observed with Moringa oleifera and Vernonia amygdalina. This effect was observed since day 1 and had the most favorable effect on day 4 and 7 for those plants. The three plants have the potential to complement existing commercial anticoccidial drugs for the control of rabbit coccidiosis and helminthiasis. However, in vivo toxicity studies of plants are recommended to investigate the potential presence of toxic effects.

Acknowledgement

The authors would like to thanks, Prof. Hounzangbé-Adoté M. Sylvie for her technical support and the technical staff at the Laboratory of Ethnopharmacology and Animal Health.

Conflicts of interest

The authors declare to have no conflicts of interest.

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