In vitro anthelmintic activity of Azadirachta indica leaf extract against Haemonchus contortus in goats

Introduction

Gastrointestinal nematode infection is the single largest health constraint facing small ruminant production in tropical and subtropical Africa, and Haemonchus contortus sits at the top of the parasite burden list [1] . Each adult female worm removes an estimated 0.05 mL of blood per day; in heavy infections exceeding 5,000 worms, the cumulative haemorrhage produces severe anaemia, hypoproteinaemia, bottle jaw, and death if untreated [2] . South African surveys indicate that clinical haemonchosis costs the small ruminant industry over ZAR 400 million annually through mortality, reduced growth rates, and treatment expenditures [3] . For decades, synthetic anthelmintics benzimidazoles, imidazothiazoles, and macrocyclic lactones formed the backbone of worm control. But repeated, often indiscriminate, use has selected for resistant parasite populations across all three drug families in South Africa's Eastern Cape, KwaZulu-Natal, and Limpopo provinces [4] . Faecal egg count reduction tests (FECRT) on communal grazing farms have returned efficacy values below 80% for albendazole and levamisole in multiple districts, falling well short of the 95% threshold that indicates adequate drug performance [5] . Multi-drug resistance where a single H. contortus population survives exposure to all available chemical classes has been confirmed on at least 14 farms in the Eastern Cape alone [6] . Ethnoveterinary medicine has re-entered the conversation as a potential source of alternative or complementary anthelmintic agents. Azadirachta indica A. Juss. (Meliaceae), commonly known as neem, is among the most widely cited medicinal plants in African and Asian livestock pharmacopoeias [7] . Neem leaves, bark, and seeds contain a complex array of limonoids (azadirachtin A and B), tannins (both condensed and hydrolysable), saponins, flavonoids, and terpenoids, several of which have demonstrated antiparasitic properties in laboratory bioassays [8] . Condensed tannins, for example, bind to the glycoprotein-rich cuticle of nematodes, disrupting motility and feeding; saponins lyse cell membranes through detergent-like action; and azadirachtin interferes with ecdysisrelated hormonal signalling [9,10] . In vitro screening platforms the egg hatch assay (EHA) and the adult worm motility assay (AWMA) allow rapid, low-cost evaluation of candidate compounds before committing to expensive in vivo dose-finding trials [11] . The EHA quantifies the capacity of a test substance to prevent embryonation and hatching of nematode eggs, while the AWMA tracks loss of motility and eventual death of adult worms immersed in graded extract concentrations. Both assays are endorsed by the World Association for the Advancement of Veterinary Parasitology (WAAVP) for preliminary anthelmintic screening [12] . Despite the widespread traditional use of neem in African goat farming, controlled In vitro data using WAAVP-compliant protocols against South African H. contortus isolates remain limited to a single published report from KwaZulu-Natal that tested aqueous not hydroethanolic extracts at only two concentrations [13] . This research aimed to fill that evidence gap by evaluating a hydroethanolic neem leaf extract across five dose levels in both the EHA and AWMA, characterising the phytochemical profile of the extract, and comparing its efficacy against an albendazole positive control.

Material and Methods Extraction Protocol

Fresh leaves of A. indica were collected from mature trees growing on the Pretoria College of Veterinary Research campus (25°44′S, 28°13′E) during February 2023, authenticated by the National Herbarium of South Africa (voucher specimen PRE-0094217), and shade-dried at ambient temperature (22-28°C) for seven days. Dried leaves were milled to a fine powder using a laboratory grinder (Retsch ZM 200, Germany) and passed through a 500 µm sieve. A total of 200 g of powder was macerated in 1,000 mL of 70% ethanol (v/v) for 72 hours with intermittent agitation every 12 hours. The macerate was filtered sequentially through muslin cloth and Whatman No. 1 filter paper. The filtrate was concentrated under reduced pressure at 40°C in a Büchi R-210 rotary evaporator until a semi-solid residue was obtained (yield: 11.8% w/w). The residue was freeze-dried (Labconco FreeZone 6, USA) and stored at -4°C in amber vials until use. Working concentrations (6.25, 12.5, 25.0, 50.0, and 100.0 mg/mL) were prepared by dissolving the lyophilised extract in phosphate-buffered saline (PBS, pH 7.4) with 1% dimethyl sulfoxide (DMSO) as co-solvent.

Material

Adult H. contortus worms and fresh faecal samples were obtained from six Boer goats (aged 8-14 months, body weight 28-36 kg) naturally harbouring mono-specific Haemonchus infections, confirmed by faecal egg count (≥1,500 eggs per gram) and larval culture identification to species level. These goats had not received any anthelmintic treatment for at least 90 days. Adult worms were collected from abomasal contents within 30 minutes of humane slaughter at a licensed abattoir in Pretoria, washed three times in warm PBS (37°C), and examined under a stereomicroscope to confirm species identity and viability (active motility). Albendazole (Sigma-Aldrich, ≥98% purity) dissolved in 1% DMSO-PBS at 0.5 mg/mL served as the positive control; PBS with 1% DMSO alone served as the negative control. The Pretoria College of Veterinary Research Animal Ethics Committee approved the protocol (Approval No. PCVR-AEC/2023-006, dated 17 January 2023).

Methods

The AWMA was conducted following Ademola et al. [11] with modifications. Ten viable adult worms (five male, five female) were placed in each well of a 24-well plate containing 2 mL of treatment solution at each of the five concentrations, plus positive and negative controls (three replicates per treatment). Plates were incubated at 37°C in a humidified atmosphere. Worm motility was assessed at 2, 4, 8, 12, 24, and 48 hours by prodding each worm with a blunt needle; absence of movement after five seconds of stimulation was recorded as death. Mortality percentage was calculated at each time point. For the EHA, fresh faeces were pooled from the six donor goats, and eggs were recovered by a modified salt flotation technique. Approximately 100 eggs were dispensed into each well of a 24-well plate with 1 mL of extract solution or control, incubated at 28°C for 48 hours, then fixed with Lugol's iodine. Hatched larvae and unhatched eggs were counted under a compound microscope (×40). Egg hatch inhibition (%) was calculated as [(control hatch ratetreatment hatch rate) / control hatch rate] × 100. Each concentration was tested in triplicate, and the experiment was repeated twice on separate days. LC₅₀ and EC₅₀ values were estimated by probit regression (IBM SPSS 28.0). Data were analysed by one-way ANOVA with Tukey's post-hoc test; significance was set at p<0.05.

Phytochemical Screening

Qualitative phytochemical screening of the crude extract was performed using standard colorimetric and precipitation reactions [14] . Tannins were detected by the ferric chloride test (dark green colour with FeCl₃) and gelatin test (white precipitate). Saponins were assessed by the froth test (persistent foam after vigorous shaking). Alkaloids were identified by Dragendorff's reagent (orange-red precipitate) and Mayer's reagent (cream precipitate). Flavonoids were confirmed by the Shinoda test (magnesium ribbon plus HCl, producing a pink-red colour). Terpenoids were detected by the Salkowski test (reddish-brown ring at the chloroformsulphuric acid interface). Glycosides were tested by the Keller-Killiani reaction. All six phytochemical groups tested positive except glycosides, which gave a weak or negative result. Total phenolic content was quantified by the Folin-Ciocalteu method and expressed as gallic acid equivalents (GAE): the extract contained 87.3±4.1 mg GAE/g dry extract. Total tannin content (determined by the vanillin-HCl assay) was 42.6±2.8 mg catechin equivalents/g dry extract.

Results

Both assays yielded clear dose-dependent responses. In the AWMA, worm mortality increased progressively with extract concentration and exposure duration. At the lowest concentration (6.25 mg/mL), only 4.3% of worms were dead at 2 hours, rising to 41.2% by 48 hours. At the highest concentration (100.0 mg/mL), mortality reached 26.3% at 2 hours and 97.8% by 48 hours. Albendazole killed 100% of worms within 24 hours at all replicates. The negative control (PBS-DMSO) produced no mortality beyond 3.3% spontaneous death at 48 hours. The phytochemical profile (Table 1) confirmed a tannin-rich extract with substantial total phenolic content. Tannins and saponins the two classes most strongly linked to nematicidal action in published literature were both abundantly present. The moderate terpenoid signal likely reflects azadirachtin and related limonoids characteristic of neem. The heatmap (Figure 1) highlights a clear concentration-time interaction. Mortality accelerated between 8 and 24 hours at concentrations ≥25.0 mg/mL, suggesting a threshold exposure period beyond which cumulative biochemical damage overwhelms the worm's repair mechanisms. At 100.0 mg/mL, near-complete kill (97.8%) was achieved by 48 hours.

Comprehensive Interpretation

Across both assays, the neem extract exhibited dosedependent anthelmintic activity that approached but did not quite match albendazole at the highest concentration tested.

The AWMA was more sensitive than the EHA LC₅₀ (14.3 mg/mL) was lower than EC₅⁐ (21.8 mg/mL) suggesting that adult worms are more vulnerable to the extract's bioactive compounds than the thick-shelled eggs. The heatmap's temporal gradient (Figure 1) and the bar chart's dose gradient (Figure 3) together establish that meaningful nematicidal effects require both adequate concentration (≥25 mg/mL) and adequate contact time (≥8 hours). This dual threshold has direct implications for any future in vivo trial design, where abomasal transit time and extract dilution by rumen fluid would reduce effective exposure.

Discussion

The LC₅⁐ of 14.3 mg/mL recorded in the AWMA positions the neem extract within the range of other tannin-rich plant extracts tested against H. contortus. Githiori et al. [15] reported LC₅₀ values of 8-18 mg/mL for aqueous extracts of Myrsine africana and Rapanea melanophloeos bark in Kenyan goat isolates. The comparable potency observed here, using a hydroethanolic rather than aqueous solvent system, suggests that the ethanol fraction extracts additional bioactives likely terpenoids and flavonoids that complement the tanninmediated effects. Condensed tannins bind to the proline-rich glycoproteins of the nematode cuticle and gut lining, creating a protein-tannin complex that disrupts feeding, motility, and egg production [9] . The high tannin content (42.6 mg CE/g) of the present extract is consistent with this being the dominant mode of action. Saponins likely contribute a secondary lytic mechanism. These amphiphilic glycosides intercalate into lipid bilayers, forming pores that increase membrane permeability and cause osmotic imbalance [10] . The observation that mortality accelerated sharply between 8 and 24 hours at mid-range concentrations may reflect the time needed for saponin pore formation to reach a critical density. Whether tannin and saponin effects are additive, synergistic, or merely parallel remains an open question that bioassay-guided fractionation could resolve.

The gap between the extract's EC₅₀ (21.8 mg/mL for EHA) and albendazole's virtually complete activity at 0.5 mg/mL highlights a practical hurdle: crude plant extracts are, by definition, dilute mixtures requiring far higher gross concentrations to deliver a pharmacologically effective dose. Translating these In vitro results into an oral dosing regimen would need to account for ruminal dilution, passage rate, and potential degradation of phenolics by rumen microflora [16] . Encapsulation strategies such as rumen-bypass alginate beads could protect the extract through the rumen and release it in the abomasum where H. contortus resides, but such formulations add cost and complexity that may offset the affordability advantage of a plant-based remedy [17] . From a resistance management perspective, the multi-target nature of a crude extract tannins, saponins, terpenoids, and alkaloids acting through different mechanisms makes it less likely to provoke the single-locus resistance mutations that have undermined synthetic anthelmintics. Integrating neem supplementation with targeted selective treatment (treating only animals above a FAMACHA threshold) could slow resistance evolution while maintaining adequate worm control in resource-limited smallholder settings.

Conclusion

The Third, the impact of neem supplementation on fecundity measured as eggs per gram reduction in faeces over 14-28 days would provide a more field-relevant endpoint than the binary alive-or-dead measure of In vitro assays. And fourth, testing the extract against confirmed multi-drug-resistant H. contortus isolates would clarify whether plant-derived anthelmintics retain activity against worms that have escaped synthetic drug pressure, a question of direct relevance to resistance management strategies.

Acknowledgement

Acknowledgements Institutional SupportThe Pretoria College of Veterinary Research provided laboratory facilities, parasitology equipment, and access to the rotary evaporator and freeze-dryer used in extract preparation.

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