Sheep are frequently infected by Haemonchus conortus. Most clinical signs related with Haemonchosis included anaemia, paleness, weakness, ventral oedema and may lead to death [1]. H. contortus causes damage to the gastric system which affects forage consumption, makes many changes in protein absorption, as well as in energy and minerals productivity [2]. This process can have a dual impact on the digestive system. It suppresses the production of gastric acid, while simultaneously elevating levels of pepsinogen and gastrin, in addition to change caused by the adult parasite in the digestive system [3].

Widespread H. contortus infections in animals are increasingly resistant to conventional anthelmintic drugs [4]. This necessitates the development of alternative approaches to tackle both adult worms in animals and their larval stages in the environment [5]. To combat rising anthelmintic resistance, reduce drug residues in livestock, and minimize environmental pollution, new strategies are crucial [6].

Commonly, the clove’s (Dianthus caryophyll) flower buds have many uses in cooking, pharmacy, perfumery and cosmetics [7]. eugenol, eugenol acetate and a- and b-Caryophyllene are the bioactive components of clove [8], which have pharmacological effects in wide spectrum (antiviral, antimicrobial and antioxidant) [9]. Many studies reported the effect of D. caryophyll aqueous and alcoholic extracts, in various concentrations, as ovicidal and anthelmintic effects on H. contortus and many other worms in vitro [10]. 

The aim of the present study was to investigate the efficacy of D. caryophyll on adult H. contortus isolated from sheep and to evaluate ultrastructural changes on cuticle of worms after in vitro contact with the extracts.

This research was conducted in September 2021 and observed in the Laboratory of Parasitology, Faculty of Veterinary Medicine, and University of Tikrit.

Extract preparation

Dried cloves of D. caryophyll were obtained from local markets which were authenticated from a Botanist of Tikrit University, College of Sciences. The cloves were oven-dried for 6 hours at a temperature of 45˚C and then grounded into fine powder. Aqueous and Ethanol extracts were prepared by cold soaking method [11]. Prepared extracts were refrigerated until use.

Worms collection and in vitro test

To study the effects on H. contortus, adult worms were collected from naturally infected sheep slaughtered in local Tikrit slaughterhouses. Following the protocol outlined by Hounzangbe-Adote et al. [12], an adult motility assay was then performed. The study tested the effectiveness of plant extracts against H. contortus at various concentrations (25, 50, 75, and 100 mg/ml). Each concentration was used in triplicate, alongside positive (bendazole) and negative (PBS) controls. Worms were incubated with the solutions at 37°C for 48 hours and observed hourly for mortality, assessed by needle pricking and 50°C water immersion.

Scanning Electron Microscopy (SEM)

At the College of Pharmacy, University of Basra, scanning electron microscopy (SEM) was used to analyze adult worms. To prepare the samples for high-resolution imaging, both treated and control worms were preserved in a 5% glutaraldehyde solution within a phosphate emulsion for 24 hours at a neutral pH of 7.4 (following a protocol by [13]. Following drying at 4°C, the samples (adult worms) underwent a crucial stabilization process at the College of Pharmacy, University of Basra. This involved three washes in a phosphate emulsion, followed by incubation in a 1% osmium tetroxide solution for 1 hour (pH 7.4). Finally, they were gradually washed, dehydrated, and dried using increasing ethanol concentrations.

Statistical analyses

Data recorded have been statistically analyzed by using PROC FREQ in SAS version 9.0

Table (1) showed gloves can kill H. contortus adult worms in vitro. The concentration 100% of the aqueous extract of carnation flowers had a significant bioactive effect on the nematodes in a period of (1.52 ± 0.1) hrs., while the concentration 25% showed the least effect compared to the negative control group Both concentrations 75 and 100% of the alcoholic extract of the same plant showed a significant effect in a period of (2.30 ± 0.09) and (1.58 ± 0.23) hrs. ,respectively, compared with the other concentrations or the negative control group, table (2).The results of the variance analysis conclude that time and dose had significant effects on mortality rates of H. contortus in vitro. 

Table (1): Effect of aquatic extract of   on adult H. contortus

Table (2): Effect of alcoholic extract of   on adult H. contortus

The SEM changes detected in the in vitro trial proved the effect of Dianthus caryophyll extracts on worm structural components. It showed deformation in the furrows and annuli of the cuticle, and longitudinal wrinkles on the outer surface of the treated worms, rupture areas also found on the outer surface of the worms compared with the control group, figure (1 & 2).

Figure (1): Ultrastructure of the cuticle of the Haemonchus sp. of negative control with SEM.

Figure (2): Ultrastructure of the cuticle of Haemonchus sp. treated by a dose of 100 mg/ mL with SEM.

Pharmacological studies detected that D. caryophyllus possessed antiviral, antibacterial, antifungal, insecticidal, antioxidant, effects [19 Phytochemical tests occurred by many studies showed that the plant contained triterpenes, alkaloids, Coumarin and cyanogenic glycoside. Many studies demonstrated the most important bioactive compounds in carnation flower oil by using gas chromatography-mass spectrometry technique (GC-MS): eugenol, phenyl ethyl alcohol, eicosene, hexyl and hexenyl benzoate, hexadecanoic acid [14-15].

Mounting evidence from multiple studies [15-17] demonstrates that eugenol possesses antiparasitic properties, effectively impacting the growth and survival of diverse parasites like Giardia lamblia, Leishmania donovani and Trypanosoma cruzi.

Tables 1 and 2 reveal the substantial mortality rate of H. contortus exposed to clove extracts. Additionally, Figure 2 showcases the scanning electron microscopy (SEM) images of cuticular damage inflicted on the worms. These findings suggest that the potent parasiticidal effect of clove extracts stems from their ability to both disrupt the cuticle and tegument of helminths and interfere with their energy metabolism through fumarate reductase and succinate dehydrogenase inhibition, ultimately dislodging the parasites from the host's abomasum [18]. The anthelmintic activity of These plant extracts have an effect against helminthes that due to the presence of metabolites with biological activity [19-20]. Tannins shows direct or indirect influence on the worm, study showed tannin direct effects on A. galli by attaching to the cuticle [21]. Studies by Zhong et al. [22] identified specific components in the plant extract with potent anthelmintic effects against H. contortus. Tannins and two flavonoids (quercetin and luteolin) were shown to effectively inhibit the growth of larval worms (L3) in vitro. Additionally, phenolic antioxidants present in the plant contribute to its worm-killing activity [23]. Another study demonstrated an increase in cell membrane permeability, where these metabolites stimulate formation of complexes with cellular membranes, causing these parasites to die [24-25]. In a study by [26], clove ethanol extract was tested for its impact on both the movement and the specific enzyme activity (acetylcholinesterase) of a parasitic flatworm known as Paramphistome cotylophoron. 

The potent worm-killing effect of clove extracts might be explained by their ability to severely damage the protective layers (cuticle and tegument) of helminths, potentially through a corrosive action. However, this mechanism requires further investigation to be fully understood.

1. Taylor, Mike A., Robert L. Coop, and Richard L. Wall. Veterinary parasitology. John Wiley & Sons, 2015.https://books.google.com/books?hl=en&lr=&id=jelbCwAAQBAJ&oi=fnd&pg=PR21&dq=Taylor+MA,+Coop+RL,+Wall+RL+(2007)+Veterinary+parasitology,+3rd+ed.+Blackwell,+Oxford.%E2%80%8F%E2%80%8F%E2%80%8F&ots=Sku-PRy1Tm&sig=JKUirzWwlNdD_xLfCui8rUJBaDQ 

2. Maciel, M. V., et al. "Ovicidal and larvicidal activity of Melia azedarach extracts on Haemonchus contortus." Veterinary parasitology 140.1-2 (2006): 98-104. https://doi.org/10.1016/j.vetpar.2006.03.007 

3. Troell, Karin, et al. "Global patterns reveal strong population structure in Haemonchus contortus, a nematode parasite of domesticated ruminants." International journal for parasitology 36.12 (2006): 1305-1316. https://doi.org/10.1016/j.ijpara.2006.06.015 

4. Raza, A., et al. "In-vitro and in-vivo anthelmintic potential of different medicinal plants against Ascaridia galli infection in poultry birds." World's Poultry Science Journal 72.1 (2016): 115-124. https://doi.org/10.1017/S0043933915002615 

5. Hördegen, P., et al. "The anthelmintic efficacy of five plant products against gastrointestinal trichostrongylids in artificially infected lambs." Veterinary parasitology 117.1-2 (2003): 51-60.https://doi.org/10.1016/j.vetpar.2003.07.027 

6. Burke, Joan M., and James E. Miller. "Sustainable approaches to parasite control in ruminant livestock." Veterinary Clinics of North America: Food Animal Practice 36.1 (2020): 89-107. https://www.sciencedirect.com/science/article/am/pii/S0749072019300544 

7. Singh, Jitender, Anupama Baghotia, and S. P. Goel. "Eugenia caryophyllata Thunberg (family myrtaceae): a review." International Journal of Research in Pharmaceutical and Biomedical Sciences 3.4 (2012): 1469-1475. https://www.researchgate.net/profile/Jitender-Singh-4/publication/310799727_Eugenia_caryophyllata_Thunberg_Family_Myrtaceae_A_Review/links/5d5eaa7c92851c3763730db0/Eugenia-caryophyllata-Thunberg-Family-Myrtaceae-A-Review.pdf 

8. World Health Organization. WHO monographs on selected medicinal plants. Vol. 2. World Health Organization, 1999. https://books.google.com/books?hl=en&lr=&id=qWP4aG-wXAQC&oi=fnd&pg=PP9&dq=World+Health+Organization.+2002.+Flos+Caryophylli,+in+WHO+monographs+on+selected+medicinal+plants,+Vol.+2.+World+Health+Organization:+Geneva.+p.+45%E2%80%9354.&ots=RNq7zXTyii&sig=Ru7WBL9Ki2gqsrZ93lkGYJRf3Mc 

9. Cortés-Rojas, Diego Francisco, Claudia Regina Fernandes de Souza, and Wanderley Pereira Oliveira. "Clove (Syzygium aromaticum): a precious spice." Asian Pacific journal of tropical biomedicine 4.2 (2014): 90-96.https://www.sciencedirect.com/science/article/pii/S2221169115301763 

10. Charitha, V. Gnani, J. Adeppa, and P. Malakondaiah. "In vitro anthelmintic activity of Syzigium aromaticum and Melia dubia against Haemonchus contortus of sheep." Indian Journal of Animal Sciences 87.8 (2017): 968-970. https://www.researchgate.net/profile/V-Charitha-2/publication/319478923_In_vitro_anthelmintic_activity_of_Syzygium_aromaticum_and_Melia_dubia_against_Haemonchus_contortus_of_sheep/links/5a0c0ecf458515e482751574/In-vitro-anthelmintic-activity-of-Syzygium-aromaticum-and-Melia-dubia-against-Haemonchus-contortus-of-sheep.pdf 

11. Jonsson, Nicholas N., and Zafar Iqbal. "Syringe test (modified larval immersion test): A new bioassay for testing acaricidal activity of plant extracts against Rhipicephalus microplus." Veterinary Parasitology 188.3-4 (2012): 362-367.https://doi.org/10.1016/j.vetpar.2012.03.021 

12. Hounzangbe-Adote, M. S., et al. "In vitro effects of four tropical plants on three life-cycle stages of the parasitic nematode, Haemonchus contortus." Research in Veterinary Science 78.2 (2005): 155-160. https://doi.org/10.1016/j.rvsc.2004.05.009 

13. Morris, J. KARNOVSKY. "A formaldehyde glutaraldehyde fixative of high osmolality for use in electron microscopy." J. cell Biol 27 (1965): 1A-149A.https://www.researchgate.net/profile/Morris-Karnovsky/publication/244955881_A_Formaldehyde-Glutaraldehyde_Fixative_of_High_Osmolality_for_Use_in_Electron_Microscopy/links/547209040cf24af340c40e48/A-Formaldehyde-Glutaraldehyde-Fixative-of-High-Osmolality-for-Use-in-Electron-Microscopy.pdf 

14. Atiyah, Khansaa Hussein, and Enas J. Kadhum. "Isolation and Identification of Phenolic Compounds from Dianthus orientalis Wildly Grown in Iraq." Iraqi Journal of Pharmaceutical Sciences (P-ISSN 1683-3597 E-ISSN 2521-3512) 30.2 (2021): 122-134. https://www.iasj.net/iasj/download/3e4aaa000eb5e2dc 

15. Machado, M., et al. "Anti-Giardia activity of Syzygium aromaticum essential oil and eugenol: Effects on growth, viability, adherence and ultrastructure." Experimental parasitology 127.4 (2011): 732-739.https://doi.org/10.1016/j.exppara.2011.01.011 

16. Abdin, Malik Zainul. "Leishmanicidal activities of Artemisia annua leaf essential oil against Visceral Leishmaniasis." (2014).https://doaj.org/toc/1664-302X 

17. Santoro, Giani F., et al. "Trypanosoma cruzi: activity of essential oils from Achillea millefolium L., Syzygium aromaticum L. and Ocimum basilicum L. on epimastigotes and trypomastigotes." Experimental parasitology 116.3 (2007): 283-290. https://doi.org/10.1016/j.exppara.2007.01.018 

18. Kumar, P., and D. K. Singh. "In vitro anthelmintic activity of Allium sativum, Ferula asafoetida, Syzygium aromaticum and their active components against Fasciola gigantica." Journal of Biology and Earth Sciences 4.1 (2014).https://agro.icm.edu.pl/agro/element/bwmeta1.element.agro-f502df6f-8dde-4b29-9c4d-5c5976deab10

19. Hoste, Hervé, et al. "The effects of tannin-rich plants on parasitic nematodes in ruminants." Trends in parasitology 22.6 (2006): 253-261. https://doi.org/10.1016/j.pt.2006.04.004 

20. Vargas-Magaña, J. J., et al. "Anthelmintic activity of acetone–water extracts against Haemonchus contortus eggs: Interactions between tannins and other plant secondary compounds." Veterinary Parasitology 206.3-4 (2014): 322-327. https://doi.org/10.1016/j.vetpar.2014.10.008 

21. Spiegler, V., E. Liebau, and A. Hensel. "Medicinal plant extracts and plant-derived polyphenols with anthelmintic activity against intestinal nematodes." Natural Product Reports 34.6 (2017): 627-643.https://doi.org/10.1039/C6NP00126B 

22. Zhong, R. Z., et al. "Effects of tannic acid on H aemonchus contortus larvae viability and immune responses of sheep white blood cells in vitro." Parasite Immunology 36.2 (2014): 100-106.https://doi.org/10.1111/pim.12092 

23. Klongsiriwet, Chaweewan, et al. "Synergistic inhibition of Haemonchus contortus exsheathment by flavonoid monomers and condensed tannins." International Journal for Parasitology: Drugs and Drug Resistance 5.3 (2015): 127-134. https://www.sciencedirect.com/science/article/pii/S2211320715300038 

24. Doligalska, M., et al. "Triterpenoid saponins affect the function of P-glycoprotein and reduce the survival of the free-living stages of Heligmosomoides bakeri." Veterinary Parasitology 179.1-3 (2011): 144-151. https://doi.org/10.1016/j.vetpar.2011.01.053 

25. Srivastava, Anubha, Ajay Kumar Gupta, and A. Rajendiran. "Phytochemical screening and in-vitro anthelmintic activity of methanolic extract from the stem bark of Plumeria rubra Linn." International Journal of Pharmaceutical Sciences and Research 8.12 (2017): 5336-5341. 10.13040/IJPSR.0975-8232.8(12).5336-41 DOI: 

26. Dhanraj, K. Manoj, and L. Veerakumari. "In vitro effect of Syzygium aromaticum on the motility and acetylcholinesterase of Cotylophoron cotylophorum." (2014). https://citeseerx.ist.psu.edu/document?repid=rep1&type=pdf&doi=9d6f7dd2787eafbcf672f7dc5a6300da98551674