Plant-plant combination: an important option in the phase of failing anthelmintics to control nematodes in small ruminants.

Abstract

The present study was designed to explore how combination phytoanthelmintic therapy can be employed to enhance livestock nematode control in goats and sheep. This was motivated by wide spread emergence of resistant varieties of nematodes and related helminths of livestock against chemical anthelmintics. Ongoing trend of selection for resistance by livestock nematodes has led to general anthelmintic failure, urging exploration and potential implementation of combination anthelmintic phytotherapy as an important option. Selected and tested plant species in the current study included Allium cepa, Aloe van balenii, Ananas comosus, Bidens pilosa, Carica papaya, Crinum macowanii, Gunnera perpensa, Nicotiana tabacum, Ricinus communis, Sarcostema viminale, Trema orientalis, Urtica dioica, Vernonia amygdalina, Zanthozylum capense, Zingiber officinale and Zizyphus mucronata. From preceding studies, anthelmintic activity of plant species in the current project have been linked to some important macro anthelmintic biochemicals and grouped as such into sub-experiments (SEPs). These included alkaloids and condensed tannins in SEP 1, flavonoids in SEP 2 and, proteases and nitrogen compounds in SEP 3. Alkaloids and condensed tannins containing plant species (SEP 1), included Aloe van balenii, Crinum macowanii, Gunnera perpensa, Nicotiana tabacum, Sarcostema viminale, Vernonia amygdalina, Zingiber officinale and Zizyphus mucronata; flavonoid containing plant species (SEP 2) comprised of Trema orientalis, Urtica dioica and Zanthozylum capense; and proteases and nitrogen compound containing species (SEP 3), consisted of Allium cepa, Ananas comosus, Bidens pilosa, Carica papaya, and Ricinus communis. In vitro studies initially tested oven-dried plant vegetative samples at 10g, 20g, and 40g equivalent crude extract in 70% ethanol, and concentrated to 100ml for efficacy on mixed nematode infected Nguni goats and Merino sheep in chapter 3. It sought to test effects of concentration, plant species, animal species, interaction between concentration and plant species, interaction between concentration and animal species, and interaction among plant species, animal species and concentration on efficacy. In SEP 1, animal species (P= 0.0107) and concentration (P= 0.0005) affected efficacy. Interaction between crude extract concentration and animal species affected efficacy (P= 0.0127). In SEP 2, concentration affected (P< 0.0001) efficacy. Animal species affected efficacy (P= 0.0046). Similarly, plant species affected efficacy (P= 0.0572). There were interactions between concentration and animal species (P= 0.0010), concentration and plant species (P= 0.0123) and among concentration, animal and plant species (p= 0.0435). In sub-study (3), animal species affected (P= 0.0004) anthelmintic efficacy. Similarly, concentration affected (P= 0.0002) anthelmintic efficacy. Additionally, interaction between animal species and concentration also affected (P= 0.0015) anthelmintic efficacy. Aloe van balenii was confirmed to exert anthelmintic activity. The following in vitro study in chapter 4 evaluated combined efficacy of plant species possessing similar macromolecule(s), in SEP 1, 2 and 3 on mixed nematode parasites of sheep. It was aimed at evaluating anthelmintic potency of plant species combination with similar macromolecules, and how these molecules relate with anthelmintic trait. Sub-experiment one had twenty one (21) combinations; SEP two, three (3) and SEP three, ten (10). Crude extract of each plant species was obtained by extracting 4 g dry matter (DM) in 70 % ethanol, and each experiment ran thrice. Expected combined efficacy computed as (a + b)/2, and simple synergy (differences between combined and expected efficacies) were also computed. Webb’s synergy was computed using Webb’s fractional product method. Alkaloids, condensed tannins and flavonoids contents were quantified and, simple and multiple regression analyses ran to determine their contribution to anthelmintic efficacy. High efficacies were observed for combined plant species of SEP 1, SEP 2, and SEP 3 but within sub-experiments were not different (P>0.05). Simple synergies were mostly positive, with means of 2.5 ± 0.67 % (SEP 1), 1.8 ± 1.19 % (SEP 2), and 2.8 ± 0.30 % (SEP 3). However, Webb’s synergy were largely negative for SEP 1, SEP 2, and SEP 3, each being lower than zero. Among plant combinations, in SEP 1, condensed tannin and flavonoid contents were different (P< 0.0001), while alkaloid contents was similar (0.3037); in SEP 2 condensed tannin (P< 0.009) and flavonoid (P= 0.0211) contents were different but alkaloid contents were similar (P= 0.07); and in SEP 3, condensed tannin contents were not different (P= 0.4312), while the alkaloid (P= 0.0135) and flavonoid contents (P< 0.0001) were different. For all these macro-molecules, there was no discernible association with anthelmintic efficacy. There was potent activity arising from combinations as exemplified by high efficacy, which in the absence of any correlation is potentially attributed to activity of all macromolecules and bioactivity of other related phytochemicals. It is suggestive of a more complex and intricate macromolecular and biochemical interaction in combinations. In the following trial in chapter 5, combinations were constituted across groups from the former. This was aimed at evaluating efficacy and synergistic effects, and additionally, contribution of alkaloids, condensed tannins and flavonoids to these parameters in vitro in sheep. Intergroup combinations were thirty two (32) for condensed tannins/alkaloids and proteases/nitrogen compounds SEP 1; 13 combinations for flavonoids and alkaloids/tannin plant species in SEP 2; and 15 combinations for proteases/nitrogen compound and flavonoid containing plant species in SEP 3. Each experiment was run thrice. Extraction of plant species was done similarly to the former in chapter 3, and dosing mode also retained, but component plant species in combined pairs were from different SEPs’. Rectal faecal grabs from sheep collected and pooled to constitute test samples (chapter 4), incubated and cultured similarly. On day 13, dosing with combined plant species crude extract at 2.5 ml with double dose concentration of each constituting pair. While some controls were moistened and others treated with 70 % ethanol to eliminate potential solvent killing effect. Larval isolation was done following Baermann technique, counting using McMaster slide on day fourteenth. Corrected mortalities were evaluated following Abott’s formula and adopted as indices of observed combined efficacies. Synergistic effects were computed following Webb’s method and alternatively simple synergy from differences between observed and expected efficacies (a + b)/2. Data was analyzed following general linear model of SAS (2000). Combined efficacies of SEP 1 related species were not different, but high, mean (95.5 ± 0.12 %). Synergistic activities were similar (P= 0.3217), with mean (-4.0 ± 0.12 %). No association occurred between any of alkaloids, condensed tannins or flavonoids with observed efficacy for SEP 1, 2 and 3. Multiple regression analysis to seek any relationship among quantified macromolecules with efficacy was not useful either for SEP 1, 2 and 3. Efficacy of combinations SEP 2 were not different (p= 0.4318). Synergistic means were not different (P= 0.2685), but negative (-5.4 ± 0.34 %). Observed efficacy of combinations in SEP 3 were similar (P= 0.5968) and high, mean (95.8 ± 0.04 %). Webb’s synergy was not different (P= 0.6264) and had mean (-3.8 ± 0.04 %). All synergistic means were negative. Crude extracts of all combinations exhibited anthelmintic activity, but could not be attributed to any specific macromolecule(s). Evidently, there is more to the active principles involved than has been examined in the current study, warranting a more detailed study in succeeding chapter 6. All selected plant species were analyzed for phytochemical composition using GC/MS, in search of anthelmintic and other related biochemicals. Four grams (4 g) dry matter (DM) of each species vegetative material was extracted in 70 % ethanol, 2 μl injected into a chromatoprobe trap, and analysed for biochemical composition. Compound identification carried out using the NIST05 mass spectral library and comparisons with retention times of chemical standards done. Where available, comparisons between calculated Kovats retention indices and those published in the literature were done. Clean chromatoprobe traps run in GC/MS as controls to identify background contamination. Compounds present at higher or similar percentages in controls were contaminants and excluded from analysis. For quantification, each peak area in each sample was quantified and converted to percentage of emission, and emitted mass in Nano grams. Phytochemicals identified belonged to aldehydes, amines, sulphur compounds, nitrogen compounds, Ketones, aliphatic acids, benzenoids, alcohols, lactones, amides, alkaloids, furans and esters. Means were determine, standard deviation, sum, minimum and maximum biochemical content in Nano grams. Reference to previous screening and related bodies of work identified and profiled phytochemicals with anthelmintic and other related biological activities. Fourty six phytochemicals had antibacterial activity, 42 antioxidant activity, 38 antifungal activity, 24 antiviral activity, and 13 anthelmintic activity. Allotment of thirteen anthelmintic related phytochemicals according to occurrence in selected plant species indicated that 2 plants had one, 6 plants had two, four plants had three phytochemicals, and 4 plants four phytochemicals. It is most plausible that anthelmintic and other related biological activities exerted by these plant species are closely linked to some phytochemical(s). The following study in chapter 7 retained and analysed identified phytochemicals in chapter 6 for their relationship with observed anthelmintic efficacy, simple and Webb’s synergies. Pearson correlation coefficient was run to explore association of phytochemical candidates with observed efficacy, simple and Webb’s synergy. Multiple regressions (using a selection option stepwise) were run to explore the influence of various phytochemicals on efficacy, simple synergy and Webb’s synergy, by conducting 10 searches to identify any of such influence. Some phytochemicals had positive influences including (benzofuran, 2,3-dihydro; pyridine, 3-(1-methyl-2-pyrrolidinyl)-, (S)-; 2 propenamide; phytol; 5-hydroxymethylfurfural; furfural) and others negative influences. Some phytochemicals selected to exerted positive influence, did so on both observed efficacy and simple synergy. Both correlation matrix and multiple regression relationships pointed to more consortia of phytochemical action. In chapter 8, this study was designed to evaluate and identify the most effective combined dose of Allium cepa and Vernonia amygdalina (COMBP1); and Ananas comosus and Carica papaya (COMBP2) at 50:50 weight for weight relative to positive control, “Zolvix” on natural nematode infected sheep in vivo. Sheep were fed 1.2 kg each of 4 % urea treated veld hay, crushed yellow maize and milled Lucerne hay in ratio 1:1:1. Treatment doses included 5 g, 10 g, 15 g, and 20 g dry matter equivalent extract in 100 ml of 70 % ethanol. Two experiments; the first, evaluated egg per gram (epg) change post treatment and the second, egg hatch and larval recovery. Fifty six Merino ewes averaging 45.0 ± 0.09 kg were weighed and initial epg count done. Both parameters used as covariates at allotting sheep to treatment doses and control of experiment one. Effects of treatment over time, and interaction between treatment and time on epg were evaluated weekly for 4 weeks. In the second trial, faecal grabs from each treatment in the former were pooled, mixed and three sub-samples of 4 g incubated and cultured for egg hatch and larval recovery post treatment on days 1, 14 and 28, relative to negative control from untreated sheep. Effects of treatment, time and, interaction between treatment and time evaluated relative to egg hatch and larval recovery. In trial one, for COMBP1, initial sheep weight were similar, whereas final weight were higher (P< 0.05). Differences between initial and final weight were similar. Epg preceding dosing and others post treatment at end of weeks’ 1, 2 and 3 were lower. Epg at end of week’s 4 were higher (P< 0.05). For COMBP2, initial sheep weight preceding treatment were similar, whereas weight post treatment were mostly similar, but partially higher for treatments 1, (5 g DM equivalent crude extract), (P< 0.05). Initial epg pre-treatment for COMBP2 were similar (P> 0.05). Mean epgs post treatment at end of weeks’ 1, 2 and 3 were lower, while that of week’s 4 were higher (P< 0.05). In trial two, egg hatch and larval recovery for COMBP1 were lower for all treatments on day 1 and 14, but oppositely higher for day 28 (P< 0.05). Mean egg hatch and larval recovery of COMBP2 for days 1 and 14 were lower, whereas that of 28 day post treatment were higher (P< 0.05). Egg hatch and larval recovery increased with time (P< 0.0219). Similarly, interaction of treatments and time resulted to higher egg hatch and larval recovery (P= 0.0496). Treatment trends for both combinations were seemingly consistent for the first two weeks post treatment. During experiment one of this project, there were differences in plant species, animal species (goats and sheep), concentration of plant species crude extract and their interactions in relation to efficacy. In vitro combination phytotherapy, pairs of plant species carrying similar and different anthelmintic macromolecules exerted potent efficacy with little or no antagonism, whereas the same macromolecules did not associate with efficacy. Interaction of biochemicals in both combinations of plant species containing similar macromolecules, and combinations involving different macromolecules would most likely have been different though. Identification of various biochemicals from GC/MS analysis linked anthelmintic and other related activities to some biochemical candidates, some of which were not macromolecules tipped initially to exert this activity. This was suggestive of a wide range of biochemical interactions among these phytochemicals leading to observed combined anthelmintic activity. Multiple regression analysis also failed to linked macromolecules to anthelmintic efficacy. In relation to efficacy vis a vis identified biochemicals, some related positively, while others related negatively, and such interactions have been associated with exercise of medicinal traits by plants. It was observed in vivo studies in sheep, that combination anthelmintic therapy of Allium cepa and Vernonia amygdalina (COMBP1); and Ananas comosus and Carica papaya (COMBP2) exerted anthelmintic activity, but most likely required a second dose following fast waning activity of the first. This will potentially sustain activity longer and improve control. It is recommended that this be done, as it is critical to advancing research in this area.