Malaria disease is characterize by a flu-like illness, accompanied by fever and chills; that may be fatal, if not treated [1]. It is caused by a protozoan parasite of the genus Plasmodium [2]. Malaria disease contributes greatly to the world disease burden and accounts for many death cases recorded in hospitals. According to a recent World malaria report released in February 2020, there were 229 million cases of malaria in 2019, up from 228 million cases in 2018 and 219 million cases in 2017 respectively [3,4]. The estimated number of malaria deaths stood at 435 000 in 2017, a similar number to the previous year. Nigeria among other African countries recorded more death cases due to malaria with about 25% malaria occurrence [1]. This is apparently an indication that malaria is highly endemic in Nigeria. This presents a severe economic and health burden to the country with about 97% of the population at risk of infection [5].

Four species of Plasmodium are recognized to cause malaria in man, this includes: Plasmodium falciparum, P. vivax, P. malariae, and P. ovale, transmitted by over 70 Anopheles species. Among the malaria parasites, P. falciparum is the most severe and blamable for 90% of all malaria deaths [6]. P. berghei have been described in African murine malaria with others being Plasmodium chabaudi, Plasmodium vinckei and Plasmodium yoelii. Due to P. berghei  ability to infect rodents and the relative ease of genetic engineering, it is thus used as a well-known model organism for the investigation of malaria in man [7]. This makes this species efficient in research programs for development and screening of anti-malarial drugs and malaria vaccines that are effective [8].

Plasmodium’s life cycle is intricate, involving many developmental stages and sites, both in the vector and the animal host [9]. Although its intricate infectious cycle presents several sites for the development of specific drugs, finding a universal drug against malaria has been a perplexing task [10]. The main problem with the presently available anti-malarial drugs is the constant resistance developed by some parasites as opined by [4, 11-14]. The mode of action of the drugs used at the present time for the treatment of some parasitic infections are yet not very known, aiding mutations and initiating drug resistance by the parasites [15-18]. Artemisinin resistance has been described by White [12] and Dondorp et al. [19] in malaria endemic areas, threatening malaria control, treatment, and eradication of global efforts. As it is arising with anti-bacterial drugs, the abuse and the mono-therapeutic use of anti-parasitic drugs is also leading to resistance, increasing preoccupation about a growing arrival of new cases and inefficacy on the treatment of recurrent cases [12, 19-21]. The utilization of natural products has delivered a potential strategy for identifying novel antimalarial drugs. The utilization of animals, fungi, bacteria and plants are important springs of biologically active substances with structural variety and novel mechanisms of action, which can possibly provide patentable products [22-25].

The skin secretions and venom of amphibians are rich sources of bioactive compounds, such as peptides, bufadienolides, alkaloids, proteins and biogenic amines, which have contributed meaningfully to human health and most pharmaceutical researches [26,27]. Yang et al. [27] maintained that the toad toxins from the parotoid or skin glands have significant therapeutic value for a good number of diseases. Against this backdrop, this study screened toad venoms for its antiplasmodial potency in Swiss albino mice infected with P. berghei.

Material and Methods

Toads Collection

Toads were collected from July to August 2019 between the hours of 0700 and 1000 from rice fields located on Latitude 8º 35´0.09ʺ E and Longitude 8º 28´18.45ʺ N beside the fence of the Take-Off Site of Federal University of Lafia, Nasarawa State, Nigeria. They were collected into a ventilated container and conveyed to the Department of Zoology Laboratory in the Permanent Site of Federal University of Lafia for extraction.

Extraction Of Crude Venom Extract From Toad

The process of extraction of toad venom was according to Gao et al. [28]. The toad was massaged and then the parotoid macro-glandules was pressed out. The toad was then released to its natural environment. The collected secretion was lyophilized and stored in a freezer at the Federal University of Lafia.

Biochemical Characterization

Determination of the quantitative and qualitative chemical components of the venom was achieved by Mass Spectrometry using a Mass spectrophotometer at the Spectral Laboratory and Services, Tudun wada Kaduna South, Kaduna State, Nigeria. The Shimadzu Fourier transform Infrared Spectrophotometer- FTIR 8400 S was used for the determination of the functional groups present in the venom. Gas Chromatography Analysis of toad venom was done using gas chromatography (Perkin-Elmer 8500). The Scanning Electron Microscope energy dispersive X-ray spectroscopy (SEM-EDS) Phenom Prox, manufactured by phenom World Eindhoven (Netherlands) was used to carry out the morphology analysis (that is; analysis of the chemical elements present in the toad venom).

Experimental Animals

Twenty-five laboratory albino mice, weighing between 14g and 37g were purchased from National Veterinary Research Institute Vom, Plateau State. All animals were fed with formulated feeds and water was administered ad libitum. The animals were allowed to acclimatize for a period of 7 days prior to their randomization into the various experimental groups.

Coding And Weighing Of Animals

The mice were marked with a permanent ink markers on different anatomical parts of the body of each animal. These include, the head (HD), back (BK), tail (TL), right leg (RL), left leg (LL), right side (RS), left side (LS), right arm (RA), left arm (LA), back strip (BKST), head and back (HDBK), head and tail (HDTL), head and right leg (HDRL), head and left leg (HDLL), head and right side (HDRS), head and left side (HDLS), head and right arm (HDRA), head and left arm (HDLA), head and back strip (HDBKST), back and tail (BKTL), back and right leg (BKRL), back left leg (BKLL), back and right side (BKRS), back and left side (BKLS), back and right arm (BKRA). The weight of each animal was measured using the electronic compact scale.

Toxicity Study

The median oral lethal dose of the toad venom was determined in mice according to Lorke [29]. The toxicity test was carried out in two phases at the in-vivo laboratory of National Institute for Pharmaceutical Research and Development (NIPRID), Federal Capital Territory, Abuja, Nigeria.

Phase 1: Nine Swiss albino mice were divided into three groups with each having three individuals. The groups were administered varying doses of 10, 100 and 1000 mg/kg respectively of the toad venom and then placed under observation for 24hours in order to monitor their behaviour as well as mortality.

Phase 2: Another set of three animals, distributed into three groups of one animal each were administered varying doses of 250, 500 and 750 mg/kg respectively of the toad venom and observed for 24hours in relation to behaviour and mortality.

Then the LD50 was calculated by the formula by Lorke [29]:

LD50=√((D_0 x D_(100 )))

Where

D0 = Highest dose that gave no mortality,

D100 = Lowest dose that produced mortality.

Grouping, Infection And Treatment Of Infected Swiss Albino Mice

Twenty-five mice were marked and infected with Plasmodium berghei obtained from NIPRID and randomly divided into five groups of five mice per group. Parasites were maintained through serial blood passage in mice wherein the mice previously infected with Plasmodium berghei and with a high parasitemia level served as the donor. Blood samples was taken from the donor and diluted with phosphate-buffered saline such that 0.2 ml injected intraperitoneally into the experimental animals contained 1 × 107 infected erythrocytes. The blood of the mice were screened for infection before inoculation with parasite.

The curative method described by Ryley and Peters [30] was used. Treatment commenced following confirmation of parasitemia in the mice. Groups 1, 2 and 3 were treated with 217 mg/kg, 173 mg/kg and 130 mg/kg respectively of crude toad venom extract whereas group 4 mice were treated with 8 mg/kg of Artemisinin Combination Therapy (Arthemeter-lumerfantrin) tablet which was crushed and diluted in distilled water before administration, while those in group 5 were infected and received no treatment (control group). Administration of treatment was done orally once daily by gavage using a mouse feeding tube for four days for the standard drug used. The parasitemia of experimental mice was establish before every treatment was administered.

Determination of Parasitemia

Blood samples were collected by bleeding the mice through the tail vein. Thin blood smears were made on clean glass microscope slides. The films were dried in air and then fixed in methanol and stained with 10% Giemsa solution [31]. The stained film was then observed under the binocular compound microscope and viewed for parasitemia. The percentage parasitemia were determined by counting the number of parasitized red blood cells (pRBC) for at least six different fields.

Percentage Parasitemia = (Parasitized RBCs)/(Total RBCs) x 100

Hematological Parameters

Using the methods described by Cheesbrough [32], the Packed Cell Volume (PCV), haemoglobin (Hb) and erythrocyte (RBC) counts were determined. These parameters were determined for each mouse before infection and after treatment. Blood samples were collected from the tail of each mouse with a heparinized capillary tube with one end sealed with plasticine.

Data Analysis

Data obtained were analyzed using R Console software (Version 3.2.2). Shapiro-wilk normality test was carried out and the data was observed to be normally distributed. One-way analysis of variance (ANOVA) was used to compare the mean parasitemia in albino mice in relation to toad venom and ACT treatments. Also, mean change in body weight and as well in haematological parameters were compared using one-way ANOVA. ANOVA test was followed by a post-hoc Tukey’s Honest Significant Difference (Tukey HSD) test where there was significant difference between the means of the treatments. The level of significance was set at P < 0.05.

Table 1: Analysis of the chemical components of toad venom crude extract using the GC-MS.