Livestock Research for Rural Development 28 (12) 2016 Guide for preparation of papers LRRD Newsletter

Citation of this paper

In vitro anthelmintic activity of aqueous extracts of five medicinal plant against eggs and the infective stage of Haemonchus contortus

Morutse Mphahlele1&2, Ana Mbokeleng Tsotetsi-Khambule3&4, Leshweni Jeremia Shai5 and Dibungi Luseba1#

1 Department of Animal Sciences, Tshwane University of Technology, Private Bag X680 Staatsartillerie Road, Pretoria West 0001, South Africa
# LusebaD@tut.ac.za
2 Department of Animal Sciences, Tompi Seleka College of Agriculture, Private Bag X9619, Marble Hall, 0450, South Africa
3 Parasites, Vectors and Vector-borne Diseases Programme, ARC-Onderstepoort Veterinary Institute, Private Bag X05, Onderstepoort 0110, South Africa
4 Department of Zoology and Entomology, University of Free State (Qwa-Qwa campus), Private Bag X13, Phuthaditjhaba 9866, South Africa
5 Department of Biomedical Sciences, Tshwane University of Technology, Private Bag X680 Acardia, Pretoria 0001, South Africa

Abstract

The aim of the study was to determine the anthelmintic activity of Cassia abbreviata, Schotia brachypetala, Senna italica, Pappea capensis and Peltophorum africanum against egg and third larval stages of H. contortus.  The bark of C. abbreviata, P. capensis and P. africanum, the root bark of S. italica and the leaves of S. brachypetala were harvested, dried at room temperature in the laboratory and thereafter extracted in hot and cold distilled water. Concentrations of 2.5, 5.0 and 7.5 mg/mL of these extracts were tested for inhibitory activity against H. contortus egg hatching and larval development.

 

Hot and cold water extracts of P. africanum inhibited the highest percentage of eggs (25 and 22%, respectively) from hatching at the lowest concentration of 2.5 mg/mL. These extracts did also inhibit 86 and 80% of larval development, respectively. The highest rates of larval mortality were recorded for P. africanum and S. italica hot water extracts (68% and 65%   within two hours at the lowest concentration and 63% and 64 % for cold water extracts.  The percentage of larval mortality increased as the concentrations increased. However, the experimental results were not solely dependent on extract concentration because all the third stage H. contortus larvae died within 72 hours irrespective of the level of concentration. This work therefore supports the use of the five experimental plant species used in the present study for the control of gastrointestinal nematodes by the Bapedi people of the Blouberg Municipality in Limpopo Province of South Africa and recommend that further studies like toxicity testing and in vivo trials be made to validate the use of these plants as an alternative to anthelmintic drugs.

Keywords: aqueous extracts, ethnoveterinary medicine, Haemonchus contortus


Introduction

Internal parasite infestations constitute a major constraint to sheep production in South Africa (Perry & Randolph 1999), with Haemonchus  contortus considered the most important of all the gastrointestinal nematodes that restrain the survival and productivity of sheep and goats owned by the rural poor in the developing world (Perry et al 2002). The control programmes for H. contortus usually result in the control of other internal worms (Kandu-Lelo 2010).

 

In Africa, commercial anthelmintics are expensive and sometimes unavailable leading to the use of poor quality or altered products (Luseba & Tshisikhawe 2012).  The misuse of these synthetic medicines has led to the development of anthelmintic resistance (Lans & Brown 1998). According to Kaplan (2004), H. contortus features prominently amongst the reports of anthelmintic resistance that has emerged in all countries of the world that produce small ruminants. In Africa, anthelmintic resistance has been reported in both the commercial and resource poor farming sectors (Tsotetsi et al 2013). This justifies an urgent need to find alternatives to synthetic drugs (Shen et al 2010) such as ethnoveterinary remedies. South Africa’s indigenous people have the knowledge of plant species with anthelmintic activity. Furthermore, because of limited availability of drugs, high cost, development of resistance, chemical residue in milk and meat, the majority of world population depends on traditional remedies (Jeyathilakan et al 2011).

 

Farmers claim that medicinal plants are more effective than pharmaceuticals for chronic pathologies (Luseba et al 2007). Luseba and Van der Merwe (2006) reported the use of ethnoveterinary medicines by Setswana-speaking people in the Madikwe area of the North West province and the Tsonga speaking people of the Limpopo Province, respectively. However, ethnoveterinary medicine has proven to be ethnical and locality-specific (Luseba & Van der Merwe 2006). Thus, research findings in other areas might not be applicable to the present study area. Therefore, the aim of this study was to investigate the anthelmintic activity of plant species used to treat parasite infections of livestock by Pedi-speaking communities in the Blouberg Municipality of the Limpopo Province, South Africa for the preservation of local knowledge by way of documenting it.


Materials and Methods

Study area
Figure 1 (a): Map of South Africa showing Limpopo
Province in red (Wikipedia 2011)
Figure 1 (b): Map of Limpopo showing Blouberg
in red (Wikipedia 2011)

 

Interviews and plant collections were conducted in the Blouberg Municipality (Figures 1 (a) and 1(b) which falls under the Capricorn District Municipality and comprises the arid sweet bushveld (Acocks 1988) in the Limpopo Province. Rainfall is 400 mm per annum and the rainy season usually extends from November to February but rainfall distribution is irregular and unpredictable. Average minimum and maximum temperatures are 12░ C and 25░ C, respectively (Mara research station, South Africa). A permit (Permit no. RB 102/13) for collection of plant materials was obtained from the Limpopo Department of Environmental affairs in Polokwane before plant specimens of each species were collected, labelled and pressed according to the methods of Fish (1999).

 

Questionnaire survey

 

Purposeful sampling of homogenous groups where participants are most likely to give good insight of the phenomenon of interest was conducted (Patton 1990). A total of 60 livestock farmers with profound knowledge on plant species used for the treatment of internal parasites were interviewed. Many rural small stock farmers in Blouberg Municipality fall outside the periphery of formal livestock markets. However, statistics show that small stock rearing remains an integral part of their everyday life and it is an important source of income (Grwambi et al 2006). Semi-structured interviews were conducted using an open-ended questionnaire between May and August 2013 which aimed at collecting data relating to tree and shrub species that are used against helminthosis by livestock farmers in the Blouberg Municipality of the Limpopo Province. Areas of discussion were guided by descriptions such as farmer’s age, gender, socio-economic profile, animal husbandry and local knowledge in animal healthcare (Luseba & Van Der Merwe 2006).  Comprehensive information of plants used by the local people such as local name, indications, method of preparation, administration and dosage to formally record the ethno-botany of the area was recorded.

 

Plant material and preparation of extracts  

 

Plants cited more than two times as having anthelmintic properties by interviewed farmers were selected for the current study (Kansonia and Ansay 1997). They were collected and thereafter authenticated by a botanist (Dr. B Egan) at the University of Limpopo. In order to have an appropriate quantity of samples for laboratory analyses, approximately 1-2 kg of fresh plant materials were collected for each plant species and from two or more shrubs or trees around Blouberg Municipality of the Limpopo province (33░47′50″S 18░27′43″E). The plant materials (leaves, bark and root-bark) were collected from October to December 2013, dried at room temperature in the laboratory and weighed frequently on a daily basis to assess the moisture content until constant weight was obtained after subsequent weighing. They were then ground to fine powder using a Macsalab mill (Model 200Lab, Eriez«, Bramley, RSA). Five grams of each powdered material was extracted in hot and cold water at 10 mL/g respectively overnight. The extracts were filtered using Whatman« No.1 filter paper (Whatman, United Kingdom) and filtrates were frozen at -80oC before drying using a Lyoquest 50 freeze dryer (Labotec, South Africa). The extracts were reconstituted in distilled water for their respective stock solutions. Then, the stock solutions were diluted to the required concentrations of 2.5, 5.0, and 7.5 mg/mL for the bioassay analyses.

 

Parasite sample collection

 

According to Kandu-Lelo (2010) the control programmes for H. contortus usually result in the control of other internal worms, hence results derived from experiments using H. contortus as a model can be applied to other nematode species. Faecal samples were collected directly from recta of adult ewes experimentally infected with H. contortus field strain. The sheep belonged to Dr. P.C Van Schalkwyk of Biozetica Agri-Source (Pty) LTD and were kept on plot A 64 Buffelshoek, Mooinooi near Rustenburg in the North West Province of South Africa (25░79′08″ S 27░55′15″ E). Collected samples were immediately transported to the Helminthology laboratory of the Agricultural Research Council, Onderstepoort Veterinary Institute in a cooler box. Faecal samples were analysed using the McMaster technique (Soulsby 1982) to confirm the presence of nematode eggs and faecal cultures prepared (Reinecke 1973) to identify/confirm the nematode genera using Van Wyk et al (2004).

 

Biological assays

 

Four assays were conducted.  In order to determine the anthelmintic activity of the prepared plant extracts, Haemonchus eggs were recovered from faeces through an egg recovery assay (Maphosa et al 2010) with some modifications.

The egg hatch assay was conducted as published by McGaw et al (2007) with some minor modifications. The larval development assay was conducted as described by Bizimenyera et al (2006).  

 

Larval mortality assay was conducted according to the method described by McGaw et al (2007) with some modifications. All live and motile L3s in each well were counted and mortality was expressed as a percentage. All tests were replicated three times.


Results

Farmers’ profile

 

The highest frequencies of trees and shrubs were cited by males compared to females as shown in Table 1. Older people aged over 40 years, of which 20 (33.3%) were females and 40 (66.6%) males, reported to know more plant species used to treat internal parasites in livestock than younger people. All in all, the number of females aged under 40 years were 5 which is 8% of the total number of participants and 25% of the female participants whereas on the other hand the number of male participants under the age of 40 were 15 which is equal to 25% of the total number of people who took part in the study and 37.5% of the male participants.

Table 1. Frequencies of trees and shrubs cited by both male and females of different age groups in Blouberg Municipality

Plant

Females

Males

Freq:
21-40 years

(%)

Freq: Above
40 years

(%)

Total
Freq.

(%)

Freq:
21-40 years

(%)

Freq: Above
40 years

(%)

Total
Freq.

(%)

Peltophorum africanum

4

44.44

7

29.1

11

33.33

6

40

25

27.77

31

29.5

Senna italica

2

22.22

4

16.66

6

18.18

1

6.67

19

21.1

20

19.05

Cassia abbreviata

1

11.11

3

12.5

4

12.12

1

6.67

12

13.33

13

12.38

Schotia brachypetala

1

11.11

4

16.66

5

15.15

1

6.67

9

10

10

9.52

Pappea capensis

1

11.11

2

8.33

3

9.09

2

13.33

8

8.88

10

9.52

Ochna pulchra

0

0

1

4.16

1

3.03

1

6.67

4

4.44

5

4.76

Capparis sepiaria

0

0

1

4.16

1

3.03

1

6.67

3

3.33

4

3.81

Mormodica balsamina

0

0

0

0

0

0

1

6.67

2

2.22

3

2.86

Jethropha zeyheri

0

0

0

0

0

0

0

0

3

3.33

3

2.86

Dicerocarym senecioides

0

0

1

4.16

1

3.03

0

0

2

2.22

2

1.9

Gymnosporia senegalensis

0

0

0

0

0

0

0

0

2

2.22

2

1.9

Punicum granatum

0

0

1

4.16

1

3.03

1

6.67

1

1.11

2

1.9

Where Freq. = Frequency; % = percentage of frequency

Table 2 shows the thirteen plant species belonging to seven families that were identified of which the plant family containing more species was the Fabaceae. The five plants that were used for biological assays were selected because according to Kansonia & Ansay 1997, there is consistency when one plant is cited for the same use by more than two respondents.

Table 2. Plants and plant parts administered for the treatment of internal parasites in animals in the Blouberg Municipality

Botanical and family names

Voucher no

Common names

Habitat

Part used

Preparation

Indication

Capparis sepiaria L. var.subglara (Oliv.) DeWolf
Capparaceae

MM0001

Moopatladi: Sep; Capper bush: Eng.

Shrub

R

Crushed and immersed in water

Immune booster, internal parasites and blood purifier

Ochna pulchra Hook.f
Ochnaceae

MM0002

Monamane:Sep; Lekker breek: Afr.

Tree

B

Crushed and immersed in water

Heartwater, internal parasites and diarrhoea

Cassia abbreviata Oliv. Subsp. bearana
Fabaceae

MM0003

Monepenepe:Sep; Sjambok pod: Eng.

Tree

B

Crushed and immersed in water

Internal parasites, blackwater fever, headache, toothache, stomach-ache and abortion agent

Punica granatum L.
Lythraceae

MM0004

Garenata: Sep; Pomegranate: Eng

Shrub

F

Sliced, dried and immersed in water

Internal parasites, stomach-ache and diarrhoea

Peltophorum africanum Sond.
Fabaceae

MM0005

Mosehla: Sep; African wattle:Eng; Huilboom: Afr

Tree

B

Crushed and immersed in water

Diarrhoea, worms in sheep and human

Gymnosporia senegalensis (Lam.) Loes.
Celasraceae

MM0006

Sephato: Sep; Rooi pendoring: Afr

Shrub

L

Dried, milled to powder and mixed with water

Heartwater, internal parasites, diarrhoea and immune booster

Dicerocarym senecioides (Klotzch) Abels.
Pedaliaceae

MM0007

Mosehlo: Sep; Devil thorn: Eng.

Forb

W

Dried, ground and mixed with water

abortion agent, retained placenta and internal parasites

Pappea capensis Eckl. Zeyh
Sapindaceae

MM0008

Morobadiepe: Sep; Jacket plum: Eng.

Tree

B

Crushed and immersed in water

Internal parasites, purgative and a cure for ringworm

Clerodendrum glaborum E. Mey. Var. glabrum
Rutaceae

MM0009

Motlhokotlhoko: Sep; Smooth tinderwood: Eng.

Shrub

L

Dried, ground to powder and mixed with water

Purgative, worms and head- ache

Schotia brachypetala Sond.
Fabaceae

MM0010

Molope: Sep; African walnut: Eng

Tree

L

Dried, milled to powder and mixed with water

Diarrhoea, worms in sheep and human

Mormodica balsamina L.
Cucurbitaceae

MM0011

Segwere sa thaba;Sep

Vine

B

Sliced and immersed in water

Internal parasites, heartwater and blood purifier.

Senna italica Mill.
Sapindaceae Fabaceae

MM0012

Morotoditshosi: Sep

Legume

RB

Crushed and immersed in water

Worms, heartwater, gallsickness, intestinal diseases, and chicken pox in humans

Jatropha zeyheri Sond.
Euphorbiaceae

MM0013

Sefapabadia: Sep; Verfbol: Afr.

Tree

BLB

Sliced and immersed in water

Worms, purgative, blood purifier

Sep=Sepedi; Eng=English; Afr=Afrikaans; B=Bark; BLB=Bulb; F=Fruit; L=Leaves; RB=Rootbark; W=Whole plant

Biological assays

 

Effect of cold and hot water plant extracts on H. contortus egg hatching

 

The highest inhibition of 22 and 25%, at the lowest concentration of 2.5 mg/mL, were recorded for P. africanum cold and hot water extracts, respectively whereas P. capensis showed the lowest inhibition percentages of all the tested plants. Generally, the egg hatch assay indicated that hot water extracts significantly inhibited (p = 0.05) the hatching of a higher number of eggs compared to the cold water extracts except for P. capensis 2.5 mg/mL and 5.0 mg/mL where there was no significant difference (p=0.05) between cold and hot water plant extracts (Table 3).

Table 3. Mean inhibition percentages for the egg hatch assay for Haemonchus contortus, using different concentrations (conc.) of crude cold and hot water extracts of five plants

Plant

Cold water

Hot water

Controls

Conc. (Mg/ml)

2.5

5.0

7.5

2.5

5.0

7.5

+

-

C. abbreviata

13.00±0.00s

19.00±0.00op

37.00±1.00g

15.33±1.52qr

21.00±0.00no

40.00±1.00f

100

0

S.brachypetala

14.66±2.08rs

23.33±1.52lm

39.00±2.00fg

17.00±2.00pq

25.66±1.52k

42.66±1.52e

100

0

S. italica

17.66±1.52p

28.66±0.57j

47.66±0.57d

20.66±0.57no

32.00±1.00i

53.00±0.00c

100

0

P. capensis

5.33±1.52u

11.00±2.00t

13.66±0.57rs

7.33±1.52u

13.00±1.00st

17.66±1.52p

100

0

P. africanum

22.00±2.00mn

31.00±2.00i

55.33±0.57b

25.00±0.00kl

34.33±1.52h

58.00±1.00a

100

0

Means with the same letter (a-u) are not significantly different (p =0.05). + = Positive; - = Negative

Effect of cold and hot water plant extracts on Haemonchus contortus larval development and viability

The results for larval development and viability are reported in Table 4. Similar to the egg hatch assay, the larval development assay indicated a linear dose related inhibitory response. The highest inhibition of larval development at the lowest concentration was observed for P. africanum with 80.00 and 86.33% for cold and hot water plant extracts respectively whereas the lowest inhibition percentage at the lowest concentration was observed for P. capensis with 66.00 and 69.00% for cold and hot water plant extracts respectively. There was a significant difference between P. africanum cold and hot water extracts while the same was observed for S. italica (p = 0.05). The positive control thiabendazole induced a 100% larval development inhibition whereas the negative control inhibited 0% of pre-infective L1 and L2 larvae from developing to the parasitic infective L3 larvae stage. The plant extracts proved to be much more effective against the infective stage larvae than with respect to the pre-infective stage larvae.

Table 4. Mean inhibition percentages for the larval development assay for Haemonchus contortus, using different concentrations (conc.) of crude cold and hot water extracts of five plants

Plant

Cold water

Hot water

Controls

Conc. (Mg/ml)

2.5

5.0

7.5

2.5

5.0

7.5

+

-

C. abbreviata

69.00±0.00k

75.00±2.00ij

87.66±1.52ef

74.33±0.57ij

83.00±2.00g

91.66±2.51c

100

0

S.brachypetala

70.33±1.52k

80.66±1.52h

90.33±1.52cd

76.00±1.00ij

86.00±0.00f

95.66±0.57b

100

0

S. italica

76.33±0.57i

86.66±0.57f

100.00±0.00a

80.00±0.00h

89.00±2.00ed

100.00±0.00a

100

0

P. capensis

66.00±2.00l

70.33±1.52k

76.00±0.00ij

69.00±2.00k

74.00±0.00j

80.00±2.00h

100

0

P. africanum

80.00±1.00h

89.00±0.00de

100.00±0.00a

86.33±1.52f

90.33±3.05cd

100.00±0.00a

100

0

Means with the same letter (a-l) are not significantly different (p =0.05). + = Positive; - = Negative

Effect of cold and hot water plant extracts on larval mortality

 

The results for the larval mortality assay with hot and cold water extracts. The larval mortality rate was dose dependent in both hot water and cold water extracts. The highest rates of mortality were recorded for P. africanum and S. italica cold water extracts, with 63% mortality and 64% mortality rates within two hours at the lowest concentration, and 68% and 65% for hot water extracts respectively. The positive control Thiabandazole« killed all the L3 larvae within 2 hours of exposure even at the lowest concentration of 2.5 mg/mL, while as expected there was no larval mortality in the negative control, which was distilled water. All the hot water plant extracts recorded the highest inhibition percentages compared to all the cold water plant extracts at all concentrations.

 

About 72 hours were needed to result in a total larval mortality irrespective of the concentration. The percentage of mortality increased as the concentrations increased; however, the experimental results were not concentration dependent because all the third stage H. contortus larvae died within 72 hours at 2.5, 5.0 and 7.5 mg/mL, respectively (Table 5). Even at the lowest concentration of 2.5 mg/mL in both cold and hot water there was no extract from all five of the tested plants that induced larval mortality of less than 50 percent. No significant difference was found to exist in all the plant extracts, and in all the three different concentrations, at the observation time of 72 hours (p = 0.05).

Table 5. Mean mortality percentages for the larval mortality assay for Haemonchus contortus, using different concentrations of crude cold and hot water extracts of C. abbreviata, S. brachypetala, S. italica, P. c apensis and P. africanum

Plant

Time
(hrs)

Cold water extracts

Hot water extracts

Controls

2.5 mg/mL

5.0 mg/mL

7.5 mg/mL

2.5 mg/mL

5.0 mg/mL

7.5 mg/mL

+

-

CA

02

57.66±1.15r

60.66±1.52q

77.66±0.57hi

57.00±2.00r

58.66±0.57r

58.33±0.57r

100

0

24

80.33±1.52g

81.00±1.00g

94.00±2.00c

65.00±2.00o

75.00±2.00j

85.00±2.00f

100

0

48

91.66±1.52d

95.00±1.00c

97.00±1.00b

87.66±1.52f

92.00±2.00d

96.33±0.57b

100

0

72

100.0±0.00a

100.0±0.00a

100.0±0.00a

100.0±0.00a

100.0±0.00a

100.0±0.00a

100

0

 

SB

02

59.00±0.00q

64.33±0.57o

70.00±1.00l

55.00±2.00s

58.00±0.00r

60.00±1.00q

100

0

24

70.33±1.52l

72.33±1.52k

80.00±1.00g

63.00±0.00p

65.66±1.52o

80.33±1.52g

100

0

48

73.00±2.00k

81.00±2.00g

86.00±1.00f

74.00±0.57j

89.00±2.00e

96.33±0.57b

100

0

72

100.0±0.00a

100.0±0.00a

100.0±0.00a

100.0±0.00a

100.00±2.00a

100.0±0.00a

100

0

 

SI

02

64.33±1.52o

67.00±0.00n

72.00±0.00k

65.33±1.52o

66.33±0.57n

74.00±0.00j

100

0

24

70.00±1.00l

71.66±0.57l

77.00±1.00hi

75.33±0.57j

77.66±2.51hi

80.00±1.00g

100

0

48

78.33±1.52h

80.00±1.00g

86.00±1.00f

81.66±1.52g

89.00±4.58e

88.00±1.00e

100

0

72

100.0±0.00a

100.0±0.00a

100.0±0.00a

100.0±0.00a

100.0±0.00a

100.0±0.00a

100

0

 

PC

02

50.00±2.00t

58.00±2.00r

60.00±1.52q

51.66±1.52t

60.00±1.00q

74.00±1.00j

100

0

24

56.00±6.08r

62.66±1.52p

69.66±0.57m

64.00±0.00o

84.33±0.57f

86.00±0.00f

100

0

48

77.00±2.00hi

79.33±0.57h

89.00±1.00e

75.66±1.52j

90.33±0.57d

98.33±0.57b

100

0

72

100.0±0.00a

100.0±0.00a

100.0±0.00a

100.0±0.00a

100.0±0.00a

100.0±0.00a

100

0

 

PA

02

63.66±1.15p

64.00±1.00o

68.66±0.57m

68.00±0.00m

68.00±1.00m

69.00±2.00m

100

0

24

74.00±1.00j

85.00±2.00f

88.00±3.00e

78.66±0.57hi

90.66±1.52d

90.00±1.00d

100

0

48

80.00±1.00g

91.00±1.00d

96.00±1.00b

83.66±1.52g

96.33±0.57b

97.00±2.00b

100

0

72

100.0±0.00a

100.0±0.00a

100.0±0.00a

100.0±0.00a

100.0±0.00a

100.0±0.00a

100

0

Where CA = Cassia abbreviata; SB = Schotia brachypetala; SI = Senna italica; PC = Pappea capensis; PA = Peltophorum africanum.+=Positive; - Negative. Means with the same letter (a-t) are not significantly different (p =0.05)


Discussion

The frequencies of plants mentioned during the survey coincide with the anthelmintic activities of the plants against H. contortus. For instance P. africanum was mentioned more often than any other plant for the treatment of worm infestations in animals, while the biological assays revealed that P. africanum was the most potent plant; on the other hand, P. capensis which was the least popular plant was not as effective as P. africanum against the H. contortus at three different stages of development. There was also no significant difference (p=0.05) on the low inhibition percentage of both cold and hot water P. capensis plant extract for egg hatch assay.

 

The results from the egg hatch assay signify that, although egg hatch inhibition was observed, not all eggs were inhibited from doing so. Egg hatch inhibition percentage for P. africanum cold water extract which proved to be the most potent plant extract which recorded 22 % at the lowest concentration and 55 % at the highest concentration whereas hot water plant extract of the same plant inhibited 25% at the lowest concentration and 58% at the highest concentration Although there was some inhibition, most eggs managed to hatch and according to Molefe el al (2012) this 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.

 

This results of the present study suggests that P. africanum and S. italica are still effective in preventing the pre-infective larvae from developing to the infective L3 larvae stage even at low concentrations. As expected, there was a significant difference between 2.5, 5.0 and 7.5 mg/mL concentrations in both hot and cold water plant extracts for egg hatch and larval development assays.

 

The most outstanding feature of the larval mortality assay results was that there was no significant difference (p=0.05) between the cold and hot water plant extracts in all the concentrations at 72 hours which could mean that the longer the worms remain in contact with the plant extracts, the lower their chances of survival.  Although the results from the present study supports the theory that P. africanum has an ovicidal effect on H. contortus eggs, no significant difference was found between P. africanum cold water extract and S. italica hot water extract; furthermore, no significant difference was established between S. brachypetala cold water extract and C. abbreviata hot water extract irrespective of the concentration (p = 0.05).

 

There was no significant difference between P. africanum 2.5 mg/mL cold water extract and S. italica at the concentration of 2.5 mg/mL hot water extract for the larval development assay as well. Similarly, there was no significant difference for egg hatch inhibition (p = 0.05) between P. africanum 2.5 mg/mL hot water extract and S. brachypetala 5.0 mg/mL hot water extract, nor between S.italica 2.5mg/mL hot water extract, P. africanum 2.5 mg/mL cold water extract and Cassia abbreviata 5.0 mg/mL hot water extract. In other words, P. africanum and S. italica were effective even at low concentrations.

 

Even though P. africanum proved to be the most potent of the five plant extracts, all these (both hot and cold water extracts) managed to inhibit further development of the free living pre-infective L1 and L2 larvae into the infective L3 larvae, and the result was 100% mortality within 72 hours. It was also evident that hot water extracts were responsible for higher rates of larval mortality in all the treatments. The efficacy of any plant extract, at the lowest concentrations, against the gastrointestinal nematodes proves the anthelmintic activity of that plant (Maphosa et al 2010). It is therefore concluded that C. abbreviata, S. brachypetala, S. Italica, P. cappensis P. africanum exhibit such an activity. It is also important to note that the survey revealed water as a common solvent used in the preparation of concoctions to be given to the animals for the treatment of internal parasites, which concurs with what Bizeminyera et al (2006) reported. The high polarity of the bio-active compounds in the plants also means that these compounds may be extractable by the polar solvents available to rural users. Molefe (2013) also reported the high effectiveness of water extracts compared to acetone extracts for the egg hatching and larval mortality assay.

 

On one extreme, P. africanum proved to be the most potent plant while on the other, P. capensis was the least potent, as determined by the highest egg hatch and larval development inhibition percentages and the highest larval mortality percentage at the lowest extract concentration of 2.5 mg/mL with minimal contact time of 2 hours between the larvae and the plant extract. On the intermediate level were S. italica, S. brachypetala and C. abbreviata, respectively.

 

It is clear that P. africanum bark and S. italica root-bark recorded high anthelmintic activity and are therefore good candidates for treatment of gastrointestinal infections. However, the mechanisms of their effectiveness still remain to be tested in vivo. Furthermore, safety and toxicity studies must be conducted in vivo to determine the minimum non-lethal concentrations needed for the treatment of nematode infections.

 

Overall, the study revealed that the hot water extracts of all tested plants were more effective than cold water extracts with respect to the egg hatch inhibition, larval development and larval mortality assays. Therefore, hot water plant extracts of P. africanum bark and S.italica root-bark could be considered as a replacement for synthetic drugs. However, S. brachypetala, C. abbreviata and P. capensis exhibit moderate anthelmintic activity; as a result, they cannot be used as a sole replacement for synthetic drugs but rather as an integrated approach to achieve sustainable parasite control in ruminant production systems (Githiori et al 2006).

 

Adamu et al (2013) stated in their report on work done on the efficacy and toxicity of thirteen plant leaf acetone extracts used in ethnoveterinary medicine in South Africa, as regards egg hatching and larval development of H. contortus, that to their surprise the aqueous extract of Markhamia obtusifolia displayed double the activity of the acetone extract. According to these authors these results indicate that the anthelminthic activity of aqueous extracts of plants which had already been investigated using organic solvents should be determined. The results of the current work are crucial in advancing towards finding long term solutions in alternative treatment for since control programmes for Haemonchus contortus usually result in the control of internal worms (Kandu-Lelo 2010).


Conclusion


Acknowledgements

The authors are grateful for the nematode eggs obtained from Dr P C Van Schalkwyk of Biozetica Agri-source Pty (LTD). The co-operation, patience and guidance of Dr B Egan and Prof P Masoko (University of Limpopo, RSA) for plant identification and grinding respectively, Agricultural Extension and Veterinary staff of Blouberg Municipality, Ms L Morey of ARC central office, Pretoria Hatfield for statistical analysis and Limpopo Department of Agriculture for financial support.


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Received 24 September 2016; Accepted 11 November 2016; Published 1 December 2016

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