In vitro anthelmintic activity of seven medicinal plants used to control livestock internal parasites in chief Albert Luthuli municipality, South Africa

Teedzai Chitura, Mulungisi Richard Shiba, David Blay Afful, Kamogelo Shai, Pfunzo Tonny Muvhali and Ana Mbokeleng Tsotetsi-Khambule¹

Department of Agricultural Economics and Animal Production, University of Limpopo, P Bag X1106, Sovenga 0727, Limpopo province, South Africa
teedzai.chitura@ul.ac.za
¹ Department of Parasites, Vectors and Vector-borne diseases, Agricultural Research Council, Onderstepoort Veterinary Institute, Private Bag X05, Onderstepoort 0110, South Africa

Abstract

The present study documents medicinal plants used to control internal parasites in livestock in Chief Albert Luthuli municipality, Mpumalanga Province, South Africa. Seven plant species: Dicerocaryum eriocarpum, Pappea capensis, Aloe ferox, Helichrysumsp, Senecio congestus, Senecio barbertonicus and Gardenia sp. were identified through structured interviews with livestock farmers.

Aqueous extracts of these plants were analysed for their effectiveness as anthelmintic through egg hatch inhibition, larval development inhibition and larval mortality assays at concentration levels of 2.5, 5 and 7.5 mg/ml. Thiabendazole®) and distilled water were used as positive and negative controls respectively. A. ferox, S. congestus and Gardenia sp were the most common plants for livestock internal parasites control. Leaves were the most used plant parts, constituting 57 %. Plant extract concentrations of 7.5 mg/ml achieved higher egg hatch and larval development inhibition than lower extract concentrations of 2.5 mg/ml for most plants. Extracts of A. ferox, S. congestus, S. barbertonicus and Gardenia sp achieved larval mortality similar to the commercial drug at concentrations of 2.5 mg/ml and 5 mg/ml after 72 hours of incubation. The study concludes that four of the selected plants used by livestock farmers in the study area are good candidates for use in controlling internal parasites.

Keywords: Aloe ferox, aqueous extracts, egg hatch, Gardenia sp, larval development

Introduction

Livestock play an important role in the socio-economic activities of South Africans. Notably, livestock serve as a source of food and income especially in the rural based communities where poverty and unemployment levels are often very high (Peacock 2005; Djoueche et al 2011; Moreki et al 2010). However, the high prevalence of livestock diseases in South Africa is a major challenge particularly to the resource limited smallholder farmers in communal areas. Dreyer et al (1999) and Tyasi (2015) reported that unlike the communal livestock farming system which can afford intensive feedlot systems, smallholder farmers commonly practice extensive feeding system through utilization of natural vegetation and this exposes livestock to internal parasites in contaminated pastures thereby making internal parasitism one of the greatest challenges among smallholder livestock farmers.

Internal parasites limit livestock productivity as they reduce fertility, cause skin irritation and suck blood, ultimately leading to death (Molefe et al 2012 ; Roeber et al 2013). Presently, gastro intestinal nematode control is largely based on repeated use of synthetic anthelmintic drugs. However, the use of synthetic anthelmintic drugs is associated with several challenges such as development of resistance to the drugs (Besier 2007; Coffey et al 2007; Sager et al 2012; Wolstenholme and Kaplan 2012; Tsotetsi et al 2013), pollution of the environment (Wall 2007), chemical residues in meat and milk (Jeyathilakan et al 2012) and high costs and low availability leading to the use of poor quality or altered products (Luseba and Tshisikhawe 2012; Maroyi 2012). In light of these challenges, novel approaches such as the use of medicinal plants play a major role in the primary health care of animals in developing countries. Indigenous South Africans have been singled among those who have the knowledge of plant species with anthelmintic activity. In this regard, South African rural smallholder livestock farmers have developed a method of controlling internal parasites through the use of indigenous plants (Bussmann et al 2011; Gebrezgabiher 2013; Luseba and Tshisikhawe 2013; Mupangwa et al 2016).

Globally, research has been conducted on plant species as alternative anthelmintic drugs to manage gastrointestinal infections in livestock and several researchers reported the use of medicinal plants to be safe, sustainable and environmentally acceptable (Erasto 2003; Shen 2010), low cost relative to synthetic drugs and the perceived better effectiveness as compared to pharmaceuticals for chronic pathologies (Luseba et al 2007) as well as the presence of a mixture of active principles that could act in synergy, yielding the anthelmintic effect and limit the development of resistance (Fouche et al 2016) . Several studies have been conducted to identify plants used for ethno veterinary medicine by smallholder farmers in the rural communities of South Africa and elsewhere (Van der Merwe et al 2001; Minja et al 2004; Luseba and Va der Merwe 2006; Sindhu et al 2010; Luseba and Tshisikhawe 2013; Adamu et al 2013 ; ul Hassan et al 2014). However, limited studies paid attention to the effectiveness of indigenous plants used to control internal parasites of livestock (Mahomoodally 2013; Tyasi and Tyasi, 2015). In the Gert Sibande district of the Mpumalanga Province of South Africa, smallholder livestock farmers in the Chief Albert Luthuli municipality still depend on traditional medicinal plants in treating their animals. However, to the knowledge of the authors, no studies have been conducted to validate the efficacy of medicinal plants used in this district. Therefore, the aim of this study was to investigate the anthelmintic activity of plant species used to treat parasite infections of livestock by Swati speaking communities of South Africa.

Materials and methods

The study was conducted in Chief Albert Luthuli municipality which is situated in Gert Sibande District of Mpumalanga Province (25.4236° S latitude and 29.4724° E longitudes by 25.3224° S latitude and 31.0824° E longitudes). Agricultural activities particularly livestock rearing is an integral part of the day to day life of the residents of Mpumalanga Province.

Questionnaire survey and plant specimen collection

Field work on plant specimen collection was undertaken during September and October 2014. During the interviews and focus group discussions, a total of 7 plants specimens belonging to five families that were more popular among the livestock farmers were collected for further identification and laboratory testing. Interviewed livestock farmers identified the plant species by their vernacular names. Information on methods of preparation of these plants for deworming livestock was gathered. All specimens were then taken to the Larry Leach Herbarium, University of Limpopo for identification. Specimens were identified as D. eriocarpum, P. capensis, A. ferox, Helichrysum sp, S. congestus, S. barbertonicus and Gardenia sp.

Sub-sampling from specimens and preparation of plant extracts

From the same specimens, sub-samples were prepared, dried, pulverized and extracted at the University of Limpopo, Animal Production Laboratory. Matured plant parts were sub-sampled as follows: D. eriocarpum (leaves), P. capensis (bark), A. ferox (leaves), Helichrysum sp (leaves and stalk), S. congestus (roots), S. barbertonicus (leaves) and Gardenia sp (pods). Specific plants parts were dried in an oven at a temperature of 50°C to a constant weight, pulverized and ground (Müller et al 2006). A 25-mesh diameter sieve was used to crush the plant parts into powder which was then preserved in airtight plastic containers for future use. Ten grams of each powdered material was extracted in distilled water over night at a concentration of 10 ml/g. All extracts were filtered using filter paper (Whatman No. 1). The water extracts were freeze- dried using a freeze dryer. Individual extracts were reconstituted in their respective solvent to give a stock solution of 50 mg/ml (Ashafa and Afolayan 2009). These were diluted to the required series of concentration of 2.5, 5.0 and 7.5 mg/ml for the bioassay analysis, using tocris dilution calculation formula C₁V₁=C₂V₂ (Stephanie 2012).

Faecal sample collection and nematode diagnostic methods

Faecal samples were collected from recta of 9 adult ewes that were infected naturally in the grazing field by H contortus (Reinecke 1973). The samples were immediately transported in a cooler box to the Helminthology Laboratory, Agricultural Research Council, Onderstepoort Veterinary Institute, South Africa. Faecal cultures were prepared according to the method described by Reinecke (1973). The McMaster technique of Soulsby (1982) was used to analyse faecal samples for the presence of nematode eggs. Confirmation of the nematode genera was carried out using procedures described by Van Wyk et al (2004). A dissecting microscope (Olympus Japan) was used for egg detection and egg per gram counts of faeces were done by counting all the eggs in the chambers of the slides multiplied by 100 (Presland et al 2005).

Egg recovery, egg hatch, larval development and larval mortality assays

Egg recovery assay was conducted as described by Maphosa et al (2009). Egg concentration was estimated by counting the number of eggs in 3 aliquots of 0.5 ml of the suspension in a microscope slide repeatedly to determine the mean number of eggs per 0.5 ml suspension. Egg hatch assay was conducted as described by McGaw et al (2007) and Bizimenyera et al (2006). Approximately 80 eggs of nematode parasites were pipetted into a 96 well microtitre plate. Each tube contained 0.5 ml of egg suspension and 0.5 ml of the plant extract under investigation at increasing concentrations (2.5, 5 and 7.5 mg/ml) reconstituted in their respective solvents. In addition, Thiabendazole® as a positive control and distilled water as a negative control were subjected to the same in vitro assays as the plant extracts that were being investigated. All tests were replicated three times. The plates were covered and incubated under humidified conditions for 48 hours at 27°C. Thereafter, a drop of Lugol’s iodine solution was added to each well to stop further hatching. The number of unhatched eggs and the first stage larvae (L₁) present per well was counted using a dissection microscope. Inhibition percentages were calculated using the formula described by Cala et al (2012).

Larval development assay was conducted as described by Bizimenyera et al (2006). Thiabendazole at 2.5, 5 and 7.5 mg/ml was added to the respective plates. Incubation of the plates was continued for 5 days, after which all the plates were examined to determine the survival of the larvae at different concentrations. All the L₃ stage larvae in each well were counted and a percentage inhibition of larval development was calculated using the formula of Cala et al (2012).

Larval mortality assay was conducted according to the method described by McGaw et al (2000) and Zafar et al (2006) with some minor modifications. In vitro cultures were prepared on faecal samples. The cultures were incubated for 7 days under humidified conditions at 27˚C and on the seventh day, the L₃ larvae were harvested from the in vitro cultures prepared and poured into a single Petri dish. Approximately 0.5 ml of L₃ were pipetted into a 96 well microtitre plate and crude extracts of the same volume were added at three different levels of concentrations (2.5, 5 and 7.5 mg/ml). Thiabendazole^® was used at the same concentrations as the plant extracts and a negative control of distilled water was tested. After the addition of the extracts, larval counts were done firstly after two hours and then daily for three days. All live and dead L₃ stage larvae in each well were counted and mortality was expressed as a percentage.

Results

Information on the plant species used to control livestock internal parasites by livestock farmers in Chief Albert Luthuli municipality, South Africa is presented in Table 1.

Egg hatch inhibition assays were carried out on the aqueous plant extracts at concentrations of 2.5 mg/ml, 5 mg/ml and 7.5 mg/ml. Thiabendazole® demonstrated a 100% anthelmintic effectiveness in all the assays performed (Figure 1 and Figure 2)

Figure 1. Egg hatch inhibition (%) of aqueous plant extracts at varying concentrations (mg/ml)

Figure 2. Larval development inhibition % of aqueous plant extracts at varying concentrations mg/ml

Larval mortality assays were carried out on the aqueous plant extracts at concentrations of 2.5 mg/ml, 5 mg/ml and 7.5 mg/ml. Thiabendazole was used as a positive control. Extracts of all plant species demonstrated larval mortality abilities that were concentration and time dependent (Table 2).

Discussion

Similar to most studies, the present study used in vitro tests to evaluate the potential of indigenous plant extracts in controlling internal parasites. This is because the toxicity of some plants is not known on animals. Although, in vitro test might not indicate the complete effectiveness of medicinal plants in vivo due to other animal factors, it is necessary to evaluate the effect these plants have on internal parasites. Findings from the present study are in agreement with those of the previous researchers who indicated a dependence on medicinal plants by smallholder farmers for treating incidents of internal parasites in their livestock (Okoli 2010; Nyahangare et al 2015). Similar to previous studies, farmers’ response in this study clearly indicated that internal parasites are a serious challenge in communal livestock production systems. Poorly managed pastures, inappropriate housing and climatic conditions in the tropics which are favourable for parasite development were cited as the major reasons for the high prevalence of internal parasites (Masika and Mafu, 2004; Mungube et al 2006; Phiri et al 2007; Mwale and Masika, 2009). Four of the seven plant species that were investigated demonstrated the ability to inhibit parasite egg hatching ability and larval development in a manner which is concentration dependent.A. ferox was the most frequently used plant followed by S. barbetonicus and Gardenia J Ellis indicating that most farmers in the study area prefer these plants and constantly use them in controlling internal parasites in agreement with the findings of Moyo and Masika (2009), Maphosa and Masika (2010), and Setlalekgomo and Setlalekgomo (2013). Trotter and Logan (1986) reported that plants that are constantly used by people in a certain area are more likely to contain bioactive substances. Mupangwa et al (2016) reported 43.4% use of A. ferox in the Kwezi and Ntambethemba villages of the Eastern Cape Province of South Africa with the plant mostly mentioned for the treatment of helminths, ticks and mites. Aqueous extracts of all fractions of A. ferox used in this study showed larvicidal effects against nematodes larvae in vitro. This finding confirms the reports by Ahmed et al (2018) that A. ferox has anthelmintic activity which is concentration dependent. Maphosa and Masika (2010) and Ahmed et al (2013) reportedin vitro larvicidal effects of aqueous extracts of A. ferox leaves on H. contortus from goats. Chauke et al (2015) reported the use of boiled leaves of the Aloe species plants in mixtures with other plant species for treatment of various diseases of livestock. The 80 % larval development inhibition at a concentration of 5mg/ml reported in our study for aqueous extracts of Aloe forex is higher compared to 48.7% reported by Ahmed et al (2013) for the same concentration and time period of incubation. A. ferox has many traditional and documented medicinal uses due to its laxative effect because of the presence of glycoside aloin (Steenkamp and Stewart 2007; Eloff and McGaw 2014). Previous studies indicated that A. ferox gel contains at least 130 medicinal agents with anti-inflammatory, analgesic, calming, antiseptic, germicidal and anti-parasitic properties among its other attributes (Melin 2009; Gurib et al 2010; Mupangwa et al 2016). The low popularity of P. capensis, D. eriocarpum and H. mill could be due to the fact that some of the respondents did not have knowledge on the preparation methods and dosages for these plants. In our study, D. eriocarpum showed weak anthelmintic properties (less than 30%) with respect to all the assays performed. Van der Merwe et al (2001) and McGaw (2007) reported no anthelmintic activities for this plant. Setlalekgomo and Setlalekgomo (2013) reported other medicinal properties of this plant species such as the treatment of retained placenta in cattle. A lot more still needs to be done to investigate the anthelmintic properties of D. eriocarpum.

Most plant extracts tested in our study showed significant concentration and time dependent effects on anthelmintic activities for all the assays that were performed in agreement with the findings by Giday et al (2009). As reported by Maphosa et al (2010), the efficacy of any plant extract, at the lowest concentrations against the gastrointestinal nematodes proves the anthelmintic activity of that plant. However, Rudin (1990), Maizels et al (1993) and Rios-dealvarez et al (2012) reported no time effect on the larvae for all the plant extracts that were investigated. These authors attributed their findings to the active constituents in plant extracts having a maximum effect on nematode parasites within 48 hours. Also, the degradation of active constituents in the extracts during testing in the assay such that there are no longer effective against third or fourth stage larvae. The possible rapid development of acute resistance or different mechanisms to overcome some of the effects of the active constituents for instance via a complex mechanism on the cuticle of the worm has also been given as a possible mechanism. In the present study, extracts of S. barbertonicus achieved 100 % egg hatch inhabitation at a concentration of 7.5 mg/ml. This contradicts the findings by Molefe et al (2012) who stated that although egg hatch inhibition was observed for all the plants that were investigated in their study, not all eggs were inhibited from hatching. Molefe et al (2012) suggested that the lack of a 100 % egg hatch inhibition might be because the egg is at this stage disseminated into the environment and protected with a thick wall, causing it to be resistant to various environmental conditions. Previous studies reported various medicinal attributes of S. barbetonicus (Semenya et al 2013). However, the antiparasitic properties of this plant species have not been extensively studied. Results from the present study prompt further research on the potential of S. barbetonicus as an anthelmintic.

From this study, it is evident that S. barbertonicus leaves and Gardenia sp pods are good candidates for treatment of gastro intestinal infection. Jangde and Bansod (2004) reported anthelmintic activity of Gardenia gummifera against H. contortus in small ruminants. In previous studies, Gardenia sp particularly G. ternifolia was identified for treatment of internal parasites (Nsekuye 1994). Girgune et al (1978) and Sunita et al (2017) reported good anthelmintic activity of essential oils of Gardenia lucida. Asteraceae have been commonly used in the treatment of various diseases since ancient times, as attested by classical literature. Members of the Asteraceae are claimed to have various properties: antipyretic, anti-inflammatory, detoxifying, antibacterial, wound-healing, antihemorrhagic, antalgic, antispasmodic, and anti-tussive, and have been considered beneficial for flatulence, dyspepsia, dysentery, lumbago, leucorrhoea, hemorrhoids, hypotension, and most importantly, some are hepatoprotective, antitumor and antiparasitic (Lin et al 2008). This study concludes that S. barbertonicus and Gardenia sp exhibit such an activity. Min and Hart (2003) postulated that the condensed tannin decrease the viability of the larval stages of the nematode parasites and also interfere with the parasite egg hatching and development to infective larval stage. However, from the previous studies, it is recommended that candidate plants be subjected to in vivo studies (following safety and toxicity studies) (Kumarashingha et al 2014). Blaxter (2011), Geary and Thompson (2001), Laing et al (2013) and Kandu-Lelo. (2012) reported some discrepancies between in vitro and in vivo studies and attributed this to the variation between the free living and parasitic nematodes in targets and/ or pathways which might relate to genomic differences and evolutionary distances between the worms.

In our study, P. capensis had a mean larval mortality percentage of 48 % at a concentration of 5mg/ml and 48hrs of incubation. This finding is different from that of Mphahlele et al (2016) who reported a mean larval mortality percentage of 96 % for P. capensis at the same concentration and incubation time. However, results for egg hatch assays showed that in our study, P. capensis had a higher egg inhibition capacity of 37.3 % at 7.5mg/ml compared to 13.7 % for the same concentration reported by Mphahlele et al (2016). Literature search does not reveal much on the anthelmintic properties of P. capensis, however, this plant species has been reported to have other medicinal properties such as aphrodisiac (whole plant) (Muthee et al 2011) as well as treatment of diarrhoea (Chauke et al 2015) However, it is evident from both studies that the anthelmintic properties of P capensis are mostly as a result of the ability of the plant to induce larval mortality. The present study only tested a single aqueous solvent for the extraction process since water is a common solvent used in the preparation of concoctions (Bizeminyera et al 2006). Molefe (2013) also reported the high effectiveness of water extracts compared to acetone extracts for the egg hatching inhibition and larval mortality assay. However, water only extracts compounds of a limited range of polarity and therefore many potentially anti-parasitic compounds could have been neglected. It has been suggested that for a particular medicinal plant, there may be a relationship between the type of extraction solvent used and the efficiency of the extraction process, as well as the level of biological activity of the extracts (Melariri et al 2012). Furthermore, it was stated from previous studies that aqueous extracts contain few compounds and have very low biological activity Eloff et al ( 2005), Kotze and Eloff (2002) thereby making them to have low or negligible anthelmintic activity Bizimenyera et al (2006) and Worku et al (2009).

Conclusions and Recommendations

• The present study revealed that the use of medicinal plants to control internal parasites of livestock is a common practice among the smallholder farmers of the Chief Albert Luthuli municipality, South Africa.
  
  
• There is a need to document information on the ethno veterinary practices of the Swati speaking people for the benefit of future generations.
  
  
• The study recommends toxicity testing and in vivo trials for the five plants that showed good anthelmintic properties.

Acknowledgements

The authors are grateful for the assistance obtained from the technical staff at the Agricultural Research Council, Onderstepoort Institute, South Africa. The guidance provided by Dr T Mandiwana- Neudani (University of Limpopo, Biodiversity Department) and Professor David Norris for financial support received during data collection activities.

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Received 28 December 2018; Accepted 10 January 2019; Published 1 February 2019

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