Ovicidal, larvicidal and adulticidal activity of black pepper (Piper nigrum L.) essential oil and tea tree oil (Melaleuca alternifolia) against Haemonchus contortus

Abstract

Haemonchosis is a worldwide helminthic disease affecting ruminants. The anthelminthic resistance has become raised. Medicinal plants are safely used as synthetic anthelmintics. Currently, the efficacy of black pepper essential oil (BPO) and tea tree oil (TTO) were in vitro evaluated against Haemonchus contortus adults, eggs and larvae at concentrations of 1.25, 2.5 and 5 mg/ml in addition to the commercially used albendazole at a concentration of 10 μg/ml. Oils were used in both normal and nanoparticles-loaded forms. Oxidative stress enzymes of worms were estimated. Scanning electron microscopy (SEM) for treated worms was done. Both normal and nanoemulsion forms of both BPO and TTO stopped the adult motility [BPO 2.5 h (hrs), NBPO 1.5 h, TTO 3 h, NTTO 1.5 h] and induced a marked decrease in the oviposition. Post treatment, the egg development and hatching were significantly (P ≤ 0.05) reduced. The damage of the egg shell, embryonal cessation and destruction of larvae occurred. Noticeable elevated antioxidant enzymes (catalase CAT, glutathione transferase GST and glutathione GSH) were found, while oxidative enzymes (lipid peroxidation LPO and nitric oxide synthase NOS) decreased. Scanning electron microscopy (SEM) for both oil-treated worms revealed anterior ends damage and several cuts associated with cuticular pores. The use of albendazole induced more or less anthelmintic and enzymatic activities with less morphological alterations of adults revealed by SEM. This study proved the marked anthelmintic potency of the BPO and TTO and their nanoemulsion forms against H. contortus rather than the widely used anthelmintic drugs.

Introduction

Haemonchus contortus is a potentially pathogenic nematode of ruminants as it gains blood of the infected animals. It induces marked anemia, hypoproteinemia, decreased body gain, humble skin coat, decreased animal production, and death in severe infections (Zajac and Conboy 2006; Gilleard 2013). Young animals are extremely expected to be infected as a result of deficient immune status and the environmental pollution (Getachew et al. 2007; Sanders et al. 2020). The epidemiological importance of Haemonchus contortus is associated with the wide geographical distribution and pathogenicity as well as global economic losses.

The resistance to antiparasitic drugs in sheep and goats is promptly increasing, perhaps due to the frequent deworming. Of various helminths showing a resistance to anthelmintics, Haemonchus contortus is the most common reported nematode worldwide (Kebede 2019). The simple biology together with the frequent worm oviposition potentiates the anthelmintic resistance (Coles 2005). Improper veterinary care actively aggravates the condition (Kaplan and Vidyashankar 2012; Lespine et al. 2012; Roeber et al. 2013; Ferreira et al. 2018). Among drawbacks of the excessive usage of anthelmintics, drug residues in consumed animal products as well as the high costs of drug administration (Albadawi 2010). Alternatively, herbal treatment with more or less higher efficacy, more safety and less expensiveness increasingly develop (Torres-Acosta et al. 2012).

The low cost and efficient methods have been purposed to control and prevent infections with gastrointestinal parasites. Among those are herbal drugs and their metabolites (Vieira 2008). The selection of plant materials with potential anthelminthic activity implies challenging several plant extracts obtained through different extraction procedures. Likewise, the anthelminthic activity of those plant extracts should be evaluated using proved tests (Hoste et al. 2015).

TTO is an essential oil, extracted from leaves of Melaleuca alternifolia plant (Huynh et al. 2012) with more than 100 volatile components (Brophy et al. 1989). The antiparasitic action is obtained by blocking acetylcholinesterases (AchE) with higher acetylcholine (Ach) levels causing paralysis of roundworms (Mills et al. 2004; Wolstenholme 2011).

The biological activity of Piper, family Piperaceae, has been recorded (Jaramillo and Manos 2001). Black pepper (Piper nigrum L.) originated in India, with Piperine component is the most bioactive with anticancer, antioxidant (Manoharan et al. 2009), analgesic, antidepressants and antibacterial (Wong and Ling 2014) action. Moreover, it stimulates pancreatic and intestinal enzymes (Wattanathorn et al. 2008; Ashokkumar et al. 2021). Due to broad spectrum antimicrobial activity, black pepper oil yields a biodegradable essential oil (EO) with a high commercial value. The current study assessed the anthelmintic activity of black pepper essential oil and tea tree oil, in both normal and nanoparticles-loaded forms, at various concentrations against H. contortus.

Materials and methods

Collection of adult worms

Adult H. contortus worms were freshly obtained from abomasa of slaughtered sheep from the municipal Beni-Suef abattoir. The worms were thoroughly washed with phosphate-buffered saline (Soulsby 1986).

Collection of H.contortus eggs and first stage larvae (L1)

Eggs achieved from intact female worms. The later were identified based on morphological features, washed several times by saline solution and incubated at 37 °C for 12–24 h then eggs centrifugated and incubated in saline for 5 min with discarding females. Other female worms were incubated at 37 °C for 12–24 h and eggs were incubated at 24 °C for 24 h, then, liberated L1 centrifugated and incubated for 5 min with discarding females.

Preparation of nanoemulsion

The formulation of nanoemulsion was a two-step process, preparations of oil/water macroemulsions by combination TT or BP oil, surfactant (Tween 80), and water, at a specific concentration of one oil to two T80 using a magnetic stirrer at a speed of 500 rpm for 10 min. The TT or BP oil concentration was 5%. Then, the emulsions were prepared by forming the respective nanoemulsions using an ultrasonicator with a 750 W input power processor (Branson Probe sonicator-Advanced model, 20 kHz, Swedesboro, NJ, USA). Each concentration was sonicated for 5, 10, and 15 min, respectively. The turbulence produced by the high-energy shockwaves disrupted the droplets. The mild heat generated was regulated by positioning the sample in a container filled with ice (Ibrahium et al. 2022).

Physicochemical characterization of oils

The physical stability of the formed nanoemulsions was characterized and controlled by physicochemical characterization tests. 270–345 nm was the wave length range at which the absorbance of the emulsion was measured using a UV–visible spectrophotometer (UV-2600, Shimadz, Japan). The zeta potential, droplet size distribution (d, nm) (analysis/volume) and polydispersity index (PDI) of nanoemulsions were measured by a zeta sizer apparatus (dynamic light scattering technique) (Nano-ZS90, Malvern, UK).

Essential oils sources and GC–MS analysis

TTO is obtained from Melaleuca alternifolia plant by steam and vacuum distillation processes (Huynh et al. 2012). BPO was purchased from AOS Products Private Limited, India. The chemical analysis of EOs was performed by gas chromatography–mass spectroscopy (GC–MS) following methods of Adams (2007) and Betts (1994).

Adult worm motility test (AWMT)

The control –ve plate contained 5 adult worms + 400 ul Tween 80 + 5600 ul saline + antibiotic solution (penicillin 4%). The control + ve comprised 5 adult worms + 400 ul Tween 80 + 5540 saline + 60 ul albendazole + antibiotic solution (penicillin 4%). For oils, 5 adult worms + 400 ul Tween 80 + 5000 saline + 600 ul oil (10%) + antibiotic solution (penicillin 4%). Petri dishes incubated at 37 °C. Adult worms, motile and immotile, were counted each 30 min. Clear worm deaths (often post 36 h) denoted negative control.

Eggs reduction relative to oils treatment

It resembles the adult worm motility test but incubation of adult treated worms for 24 h, then after 24 h, the mean of deposited eggs was calculated as the equation.

$$\text{ER}\left(\text{egg reduction}\right)\%=\frac{\text{eggs of control}-\text{ve}-\text{eggs of treated}}{\text{eggs of control}-\text{ve}}\times 100$$

Egg hatching test (EHT)

The protocol of Katiki et al. (2011) was followed. Briefly, A control negative plate with 30 ul eggs (aqueous solutions of 100 eggs) + 400 ul Tween 80 (3%) + 4970 ul saline + 600 ul saline, albendazole-treated plate was used as a control positive; 30 ul eggs (aqueous solutions of 100 eggs) + 400 ul Tween 80 + 5510 saline + 60 ul albendazole, and for oils, each plate had 30 ul eggs (aqueous solutions of 100 eggs) + 400 ul Tween 80 + 5270 saline + 300 ul oil (5%). Each essential oil was applied at concentrations of 5, 2.5, 1.25 mg/ml. Post incubation at 27 °C for 24 h, eggs (hatched, not hatched and undeveloped) were counted microscopically. Morphological alterations occurred in the treated eggs were recorded.

Effect of various treatments on H.contortus first stage larvae

The same protocol as in EHT, but larvae were used instead of eggs. Morphological alterations of treated L1 were recognized.

Larval development test (LDT)

A similar protocol was done. A control negative group had 30 ul eggs (aqueous solutions of 100 eggs) + 400 ul DMSO (1%, v/v) + 5570 ul saline + nutritive medium of amphotericin B, E. coli and yeast extract. Control positive group included 30 ul eggs (aqueous solutions of 100 eggs) + 400 ul DMSO (1%, v/v) + 5510 saline + nutritive medium of amphotericin B, E. coli and yeast extract + 60 ul albendazole. For oils, 30 ul eggs (aqueous solutions of 100 eggs) + 400 ul DMSO (1%, v/v) + 4970 saline + nutritive medium of amphotericin B, E. coli and yeast extract + 600 ul oil (10%). Six days later, all larval stages were counted microscopically. The revealed findings were expressed as the larval development inhibition.

Scanning electron microscopy (SEM)

Worms were fixed with 2% glutaraldehyde solution with sodium cacodylate buffer (0.1 M) at 4 °C for 4 h. Worms washed twice in the same buffer (0.2 M), dehydrated in a graded ethanol concentrations, dried by critical point drying with EMSCOPE CPD 750, and coated with gold–palladium at 100 Å/min for 5 min. Then, worms were examined with S450 scanning electron microscope (Hitachi) at an accelerating voltage of 15 kV (Martínez-Ortíz-De-Montellano et al. 2013).

Determination of oxidative stress markers and antioxidant defense system

Catalase (CAT) activity

Spectrophotometric analysis of catalase was performed using a kit purchased from the Biodiagnostic, Giza, and Egypt Company. When exposed to a certain amount of H₂O₂, catalase reacts. With the aid of catalase, the process was stopped after exactly one minute.2H₂O₂ CAT 2H₂O + O₂.

In the presence of peroxidase (HRP), the remaining H₂o₂ reacts with 3, 5-Dicholoro- 2 hydroxybenzene sulfonic acid (DHBS) and 4-aminophenazone (AAP) to form choromophore with a color intensity inversely proportional to the amount of catalase in the original sample. 2H₂O₂ + DHBS + AAP + HRP Quinoneimine Dye + 4H₂O (Aebi 1984).

Estimation of glutathione-s-transferase (GST) concentration

Spectrophotometric analysis was used to assess glutathione-s-transferase using a kit from the Biodiagnostic, Giza, and Egypt Company. The conjugation of 1-chloro-2, 4-dinitrobenzene (CDNB) with reduced glutathione is used by the Biodiagnostic GST Assay Kit to evaluate total GST activity (cytosolic and microsomal). A rise in absorbance at 340 nm is observed in conjunction with conjugation. The rate of growth is inversely correlated with the sample's GST activity, which converts GSH into GSSG/min at 30 °C and pH 7.4 (Habig and Jakoby 1981).

Glutathione (GSH) concentration

The process is based on the appearance of a yellow colour after the addition of DTNB to molecules with sulfhydryl groups. 500 mL of phosphate buffer containing supernatants were mixed with 3 mL of 4% sulfosalicylic acid. The mixture was centrifuged for 15 min at 1600 g. 500 mL of supernatants were collected and added to Ellman's reagent. After 10 min, the absorbance at 412 nm was measured. In terms of nmol/mg tissue, the GSH concentration was expressed (Jollow et al. 1974).

Lipid peroxidation (LPO) level

Draper and Hadley (1990) used spectrophotometry to assess the index of lipid peroxidation (LPO). In a nutshell, supernatant combined with 1 mL 5% tricholoro acetic acid (TCA), centrifuged at 2500 g for 10 min, and the result was measured. 500 L of the supernatant were mixed with 1 mL of thiobarbituric acid (TBA) reagent (0.67%) before being heated at 90 °C for 15 min. After cooling, the mixture's absorbance at 532 nm was measured. Tetraethoxypropane was used to calculate the MDA values, which were then represented as nmol MDA/100mg/hr.

Estimation of nitric oxide (NO) assay

Nitric oxide was measured spectrophotometrically using kit obtained from, Biodiagnostic, Giza, and Egypt Company. In acid medium and in the presence of the nitrite the formed nitrous acid diazotize sulphanilamide and the product are coupled with N- (1-naphthyl) ethylenediamine. The resulting azo dye has a bright reddish–purple color which can be measured at 540 nm (Montgomer and Dymock 1961).

Statistical analysis

The obtained data are introduced as mean ± standard error. Differences between means in the different groups were tested for significance by one-way analysis of variance (ANOVA) and Duncan’s multiple range tests to detect the significance among means in between different experimental groups. SPSS (version 16) computer program was used (P < 0.05).

Results

H.contortus worm motility relative to the use of BPO and TTO

The examination of untreated worms revealed normal motility during the first 4 h of the experiment. Two hours later, it was moderate (+ +). It reduced and scored as mild ( +) from the 7th hour till 24 h from the beginning of the application. In albendazole-treated groups, normal motility was noticed during the first 2 h. After 2.5 h, the motility was moderate and still for 3 h, then, it declined and expressed as mild till 4.5 h. A complete cessation of motility occurred post 4.5 h (–). In NBP 5%-treated groups, the movement was moderate after half an hour. One hour later, it was mild and completely disappeared after one and half hour from the beginning of the application. In NBP 2.5%-treated groups, a marked motility was seen by naked eyes during the first 30 min. One hour later, it became moderate, then, it declined as mild till 2 h. The motility completely disappeared after 2 h. Meanwhile, in NBP 1.25%-given worms, a normal motility was recorded during the first 60 min, became a moderate at the 2nd hour. It reduced and scored as mild till 3 h post application then the movement completely stopped. Similarly, in BP 5%-treated groups, a normal motility was recorded during the first 30 min. One hour later, it was moderate and it reduced and scored as mild till 2 h. The motility disappeared after 2.5 h. In BP 2.5%-treated worms, a normal motility was recorded during the first 60 min. After 1.5 h, the motility was moderate and remained moderate till 2.5 h, then, it declined and expressed as mild till 2.5 h. The motility disappeared after 4 h. Moreover, in BP 1.25%-given worms, a normal motility was recorded during the first 2.5 h, then it was moderate at the 2nd hour till the 3rd hour post application. The movement reduced and scored as mild till 4.5 h post application then the movement completely stopped. Similarly, In NTT 5%-treated groups, the movement was moderate after half an hour and one hour later, it was mild and completely disappeared after one and half hour from beginning of the application. In NTT 2.5%-treated groups, a normal motility was recorded during the first 30 min. After one hour, it was moderate till 2 h, then, it declined and expressed as mild till 2.5 h. A complete cessation of motility occurred after 2.5 h. Meanwhile, worms treated with NTT 1.25% showed a normal motility was determined during the first 1.5 h, then, it was moderate after the 2nd hour and it declined and expressed as mild till 3 h, then it completely stopped. In TT 5%-treated groups, a normal motility was recorded during the first 30 min. One hour later, it was moderate, reduced, scored as mild till 2.5 h and disappeared after 3 h. In TT 2.5%-treated groups, a normal motility was recorded during the first 2 h. After 2.5 h, the motility was moderate till 3 h, then, it declined and expressed as mild till 4.5 h and disappeared after 4.5 h. At the concentration of 1.25%, a normal motility was detected during the first 2 h, then, it was moderate at the 2.5 h till the 4th hour post application. The motility declined and expressed as mild till the 5th hour post application and completely stopped (Table 1).

Table 1.

Motility findings of H.contortus adult worms exposed to various treatments

	Time post treatment (hours)
	Zero	1/2 hr	1 hr	1.5 hrs	2 hrs	2.5 hrs	3 hrs	3.5 hrs	4 hrs	4.5 hrs	5 hrs	5.5 hrs	6 hrs
Control -ve	+++	+++	+++	+++	+++	+++	+++	+++	+++	++	++	++	++
Albendazole	+++	+++	+++	+++	+++	++	++	+	+	+	_	_	_
NTT 5%	+++	++	+	_	_	_	_	_	_	_	_	_	_
NTT 2.5%	+++	+++	++	++	++	+	_	_	_	_	_	_	_
NTT 1.25%	+++	+++	+++	+++	++	+	+	_	_	_	_	_	_
TT 5%	+++	+++	++	++	+	+	_	_	_	_	_	_	_
TT 2.5%	+++	+++	+++	+++	+++	++	++	+	+	+	_	_	_
TT1.25%	+++	+++	+++	+++	+++	++	++	++	++	+	+	_	_
NBP 5%	+++	++	+	_	_	_	_	_	_	_	_	_	_
NBP 2.5%	+++	+++	++	+	+	_	_	_	_	_	_	_	_
NBP 1.25%	+++	+++	+++	++	++	+	+	_	_	_	_	_	_
BP 5%	+++	+++	++	+	+	_	_	_	_	_	_	_	_
BP 2.5%	+++	+++	+++	++	++	++	+	+	_	_	_	_	_
BP 1.25%	+++	+++	+++	+++	++	++	++	+	+	+	_	_	+

 +  +  + denotes clear macroscopic motility +  + denotes moderate motility + denotes mild motility–denotes paralysis

Effect of BPO and TTO on the oviposition

The mean of oviposition was significantly (P ≤ 0.05) decreased in all treated worms. In albendazole-treated groups, the egg reduction was 94.7%, while in NBP 5.0%, NBP 2.5% and NBP 1.25%-treated worms, it decreased to s97.9, 77.9 and 67.1%, respectively. Similarly, in BP 5.0%, BP 2.5% and BP1.25%-treated worms, it was 93.8, 79 and 59.5%, respectively. Regarding NTT 5.0%, NTT2.5% and NTT1.25%-treated worms, it was 95.4, 76.9 and 60.5%, respectively. In TT 5.0%, TT 2.5% and TT1.25%-treated worms groups, it was 81, 61.5 and 32.8%, respectively (Table 2).

Table 2.

Fecundity and egg reduction of the treated worms

Drug/conc	Mean of egg deposition ± S.E	Egg reduction % (efficacy %)
Control -ve	195,000 ± 5477.23a	Zero
Albendazole	10,360 ± 554.83e	94.7
NTT 5%	9000 ± 1095.45e	95.4
NTT 2.5%	45,000 ± 2898.28d	76.9
NTT 1.25%	77,000 ± 4427.19c	60.5
TT5%	37,000 ± 2280.35d	81
TT 2.5%	75,000 ± 5477.23c	61.5
TT 1.25%	131,000 ± 3521.36b	32.8
NBP5%	4000 ± 632.46e	97.9
NBP2.5%	43,000 ± 1673.32d	77.9
NBP1.25%	64,000 ± 3346.64c	67.1
BP5%	12,000 ± 1095.45e	93.8
BP2.5%	41,000 ± 2280.35d	79
BP1.25%	79,000 ± 1673.32c	59.5

Superscript of the same letter in cells of the same column is non-significant. Superscript of different letters in the same column is significant (P ≤ 0. 05).

Development and hatching of H. contortus eggs post exposure

The egg development and hatching were significantly (P ≤ 0.05) reduced in all treated groups. In the control untreated group, the mean of hatched eggs was 85.67 ± 2.20, and in the developed not hatched eggs, it was 9.67 ± 0.91 but that of undeveloped eggs was 4.67 ± 1.28. In albendazole-treated groups, there was a significant decrease in the hatched eggs and increase in that of the undeveloped eggs. The means were 7.00 ± 1.32, 7.67 ± 0.42 and 85.33 ± 0.92, respectively. Meanwhile, in NBP 5.0%-treated eggs, they were 2.33 ± 0.56, 9.33 ± 0.21 and 88.33 ± 0.56, respectively. For NBP 2.5%-treated eggs, means were 6.33 ± 0.76, 8.67 ± 0.56 and 85.00 ± 0.97 for hatched, developed not hatched and undeveloped eggs. Similarly, in NBP1.25%-treated eggs, means were15.67 ± 0.92, 20.33 ± 1.73 and 67.33 ± 2.35.

Similarly, in BP 5.0%-treated eggs, they were recorded as 8.00 ± 0.37, 18.00 ± 1.59 and 74.00 ± 1.26, respectively. For BP 2.5%-treated eggs, means were 15.67 ± 0.56, 22.33 ± 2.20 and 62.00 ± 2.39 for hatched, developed not hatched and undeveloped eggs. In addition, in BP1.25%-treated eggs, means were 10.33 ± 1.05, 26.00 ± 1.32 and 60.67 ± 4.90. Meanwhile, in NTT 5.0%-treated eggs, they were 4.00 ± 0.37, 7.00 ± 0.73 and 89.00 ± 0.97, respectively. For NTT 2.5%-treated eggs, means were 6.67 ± 0.76, 11.67 ± 0.56 and 81.67 ± 0.92 for hatched, developed not hatched and developed not hatched eggs. Similarly, in NTT 1.25%-treated eggs, means were 15.67 ± 0.92, 20.33 ± 1.73 and 67.33 ± 2.35. Meanwhile, in TT 5.0%-treated eggs, they were 5.67 ± 0.92, 9.33 ± 0.56 and 85.00 ± 0.37, respectively. For TT 2.5%-treated eggs, means were 10.33 ± 0.56, 15.33 ± 1.65 and 74.33 ± 1.87 for hatched, developed not hatched and undeveloped eggs. Similarly, in TT1.25%-treated eggs, means were 26.67 ± 1.52, 17.33 ± 0.56 and 56.00 ± 1.5 (Table 3).

Table 3.

Means of development and hatching of H. contortus eggs post exposure

Drug/conc	Means of hatched eggs ± S.E	Means ofdeveloped not hatched eggs ± S.E	Means of undeveloped eggs ± S.E
Control -ve	85.67 ± 2.20a	9.67 ± 0.92f	4.67 ± 1.28a
Albendazole	7.00 ± 1.32c,d,e	7.67 ± 0.42f	85.33 ± 0.92f
NTT 5%	4.00 ± 0.37d,e	7.00 ± 0.73f	89.00 ± 0.97f
NTT 2.5%	6.67 ± 0.76c,d,e	11.67 ± 0.56e,f	81.67 ± 0.92e,f
NTT 1.25%	9.67 ± 0.56c	12.00 ± 0.37d,e,f	75.00 ± 1.32d,e
TT5%	5.67 ± 0.92c,d,e	9.33 ± 0.56f	85.00 ± 0.37f
TT 2.5%	10.33 ± 0.56c	15.33 ± 1.65c,d,e	74.33 ± 1.87d,e
TT 1.25%	26.67 ± 1.52b	17.33 ± 0.56b,c,d	56.00 ± 1.59b
NBP5%	2.33 ± 0.56e	9.33 ± 0.21f	88.33 ± 0.56f
NBP2.5%	6.33 ± 0.76c,d,e	8.67 ± 0.56f	85.00 ± 0.97f
NBP1.25%	15.67 ± 0.91c	20.33 ± 1.73b,c	67.33 ± 2.35c,d
BP5%	8.00 ± 0.37c,d	18.00 ± 1.59b,c	74.00 ± 1.26d,e
BP2.5%	15.67 ± 0.56c	22.33 ± 2.20a,b	62.00 ± 2.39b,c
BP1.25%	10.33 ± 1.05c	26.00 ± 1.32a	60.67 ± 4.90b,c

Superscript of the same letter in cells of the same column is non-significant. Superscript of different letters in the same column is significant (P ≤ 0. 05).

Larval development of H.contortus post exposure

The development of H.contortus eggs in the control untreated group was 80.0%. Meanwhile, in all treated groups, the development was recorded zero (no L3 developed).

Effect of various treatments on H.contortus eggs and first stage larvae

In albendazole-treated group, eggs elucidated cessation of embryonation as well as destructive embryos. Moreover, larvae had a destructive cuticle with extrusion of internal contents (Fig. 1c, d).

Fig. 1.

Albendazole and control untreated eggs and L1 of Haemonchus contortus. a Control untreated eggs. Note oval egg with normal intact shell, with normal embryogenesis. Scale bare 50 µm. b control untreated larva. Normal intact cuticle and internal contents were seen. Scale bare 100 µm. c Albendzole treated-eggs showing cells with cessation of embryonation and damaged embryos. Scale bare 50 µm. d Albendzole treated-larva. Note a damaged cuticle with extrusion of internal contents. Scale bare 100 µm

In NBPO-treated group, morphological alterations ranged from cessation of embryonation, more or less degenerative changes of embryos, some destructive changes of shells and remarkable larval cuticular destruction associated with rupture of internal organs (1.25%) (Fig. 2a, b). Eggs and larvae treated with NBPO 2.5% showed similar alterations with clear embryonal shrinkage (Fig. 2c, d). Moreover, in NBPO 5.0%-treated eggs/larvae, additional deformity of egg shells shapes (spindle rather than oval) occurred (Fig. 2e, f). Meanwhile, BPO-treated eggs/larvae had similar changes with initial destruction of the cuticle particularly at the anterior end of the larvae (1.25%) (Fig. 2g, h), mild destructive changes in egg shells/larval cuticle (2.5%) (Fig. 2i, j) and severe damage in eggs/larvae treated with a concentration of 5.0% (Fig. 2k, l). In NTTO and TTO-treated group, similar morphological alterations revealed (Fig. 3a–l).

Fig. 2.

NBPO- and BPO-treated eggs and L1 of Haemonchus contortus. a NBPO 1.25%-treated eggs. Cessation of embryonation, more or less degenerative changes of embryos and some damage in shell were found. Scale bare 100 µm. b NBPO 1.25%-treated larva. Note a damaged cuticle with extrusion of internal contents. Scale bare 100 µm. c NBPO 2.5%-treated egg. Stopped embryonation with shrinkage of embryos and some damage of shell were noticed. Scale bare 100 µm. d NBPO 2.5%-treated larva revealing damaged cuticle with extrusion of internal contents. Scale bare 50 µm. e NBPO 5%-treated eggs with severe deterioration of shell, cessation of embryonation and morphological alterations. Scale bare 50 µm. f NBPO 5%-treated larva with a severe damaged cuticle with extrusion of internal contents. Scale bare 50 µm. g BPO 1.25%-treated egg. Note cells with cessation of embryonation with more or less damage of embryo and the shell. Scale bare 50 µm. h BPO 1.25%-treated larva. An initial destruction of the cuticle anteriorly with extrusion of internal contents was noticed. Scale bare 100 µm. i BPO 2.5%-treated egg. Note a cessation of embryonation with destruction of the embryo and some damage of the shell. Scale bare 50 µm. j BPO 2.5%-treated larva revealing damaged cuticle with extrusion of internal contents. Scale bare 100 µm. k BPO 5%-treated egg. Severe damage of shell with cessation of embryonation and degenerative changes of the embryonal cells were detected. Scale bare 50 µm. l BPO5%-treated larva with a severe damaged cuticle with extrusion of internal contents. Scale bare 100 µm

Fig. 3.

NTTO and TTO-treated eggs and L1 of Haemonchus contortus. a NTTO 1.25%-treated eggs. Note cells with cessation of embryonation, degenerative changes of embryos and the shell. Scale bare 100 µm. b NTTO 1.25%-treated larva with damaged cuticle and extrusion of internal contents. Scale bare 100 µm. c NTTO 2.5%-treated egg with stopped embryonation, damage of the shell. Scale bare 50 µm. d NTTO 2.5%-treated larva with damaged cuticle and extrusion of internal contents. Scale bare 50 µm. e NTTO 5%-treated egg with severe damaged shell and stopped embryonation. Scale bare 50 µm. f NTTO 2.5%-treated larva with severe damaged cuticle. Scale bare 50 µm. g TTO 1.25%-treated egg with a normal shell and stopped embryonation. Scale bare 100 µm. h TTO 1.25%-treated larva with an initial damaged cuticle anteriorly and extrusion of internal contents. Scale bare 100 µm. i TTO 2.5%-treated egg with stopped embryonation, damaged embryo and egg shell. Scale bare 50 µm. j TTO 2.5%-treated larva with a damaged cuticle and extrusion of internal contents. Scale bare 100 µm. k TTO 5%-treated egg with severe damaged shell and stopped embryonation as well as damaged embryonal cells. Scale bare 100 µm. l TTO 5%-treated larva with severe damaged cuticle and extrusion of internal contents. Scale bare 100 µm

Characterization of nano-emulsion forms of oils, NTTO and NBPO

The average particle size distribution of TTO was 157.5 nm, with PdI of 0.560 and zeta potential 0.00548 Mv. While, the average particle size distribution of BPO was 168.0 nm, with PdI of 0.499, and zeta potential 0.341Mv. The wave length of TTO was 241 nm at 0.064 absorbance, for BPO it was 226 nm at 0.711 absorbance.

Chromatography–mass spectroscopy (GC–MS) of NTTO and NBPO

According to GC–MS analysis, piperine was the main constituent of BPO (50.0%).While gamma-terpinene was the main component of TTO (32.92%).

Determination of oxidative stress markers and antioxidant defense system

Activation of catalase against ROS generated stress

The enzymatic activity was calculated. A steady increase in CAT activity when exposed to a drug treatment with an increase of stress response synchronized with an increase of oils concentration had been observed. In the control untreated groups, it was 0.26 µ/mg, while in albendazole-treated groups, it reached 1.81 µ/mg. For NBP 5.0%, NBP 2.5% and NBP 1.25%, it was 1.37 µ/mg, 1.35µ/mg and 0.39 µ/mg, respectively. Meanwhile, for BP 5.0%, BP 2.5% and BP 1.25%, it was 0.99 µ/mg, 0.05 µ/mg and 0.13 µ/mg, respectively. Similarly, for NTT 5.0%, NTT 2.5% and NTT 1.25%, it was 0.58 µ/mg, 0.25 µ/mg and 0.27 µ/mg, respectively. In groups treated with TT 5.0%, TT 2.5% and TT 1.25%, it was 0.72 µ/mg, 0.29 µ/mg and 0.21 µ/mg, respectively (Table 4 and Fig. 4).

Table 4.

Determination of oxidative stress markers and antioxidant defense system of adult H. contortus worms after treatment with various oils

Groups	Catalase (µ/mg tissue)	GSH (nmol/100 gm tissue)	GST (µ/g tissue)	LPO (nmol/100 mg tissue/hour)	Nitrate (µ/mg tissue)
control−ve	0.26 ± 0.00017a	31.00 ± 0.03a	337.27 ± 1.38a	2.40 ± .01h	0.26 ± 0.00436a
Albendazole	1.81 ± 0.00751b	91.38 ± 0.36h	891.26 ± 2.72e	0.82 ± 0.10b	0.13 ± 0.00436c,d
NTT 5%	0.58 ± 0.00028g	84.74 ± 0.17g	822.52 ± 1.36d,e	0.63 ± 0.05a	0.17 ± 0.00490b
NTT 2.5%	0.25 ± 0.00028j	79.61 ± 0.10f	789.48 ± 19.46d,e	1.23 ± 0.01e	0.13 ± 0.00585c,d
NTT 1.25%	0.27 ± 0.00022a	70.35 ± 0.53b,c	722.52 ± 20.68c,d	1.91 ± 0.01g	0.19 ± 0.00558b
TT5%	0.72 ± 0.00110f	77.64 ± 2.67e,f	817.62 ± 1.80d,e	1.04 ± 0.02c,d	0.13 ± 0.00585c,d
TT 2.5%	0.29 ± 0.00110i	72.41 ± 0.48d	653.19 ± 1.81c	1.03 ± 0.02c,d	0.19 ± 0.00558b
TT 1.25%	0.21 ± 0.00097k	69.77 ± 0.45b,c	518.65 ± 3.05b	0.89 ± 0.03b,c	0.18 ± 0.00558b
NBP5%	1.37 ± 0.00232c	96.73 ± 1.20i	814.64 ± 37.76d,e	0.65 ± 0.06a,b	0.15 ± 0.00365c
NBP2.5%	1.35 ± 0.00117d	80.59 ± 0.71f,g	730.42 ± 19.18c,d	1.65 ± 0.02f	0.18 ± 0.00365b
NBP1.25%	0.39 ± 0.00018h	74.95 ± 0.12d,e	796.95 ± 2.69d,e	1.80 ± 0.04f,g	0.15 ± 0.00365c
BP5%	0.99 ± 0.00072e	78.19 ± 0.45e,f	795.70 ± 28.77d,e	1.10 ± 0.02d,e	0.12 ± 0.00558d
BP2.5%	0.05 ± 0.00011m	77.64 ± 0.47e,f	638.95 ± 26.08b,c	1.12 ± 0.01d,e	0.15 ± 0.00365c
BP1.25%	0.13 ± 0.00010l	67.33 ± 0.32b	635.15 ± 75.45b,c	1.68 ± 0.00557f	0.18 ± 0.00365b

Superscript of the same letter in cells of the same column is non-significant. Superscript of different letters in the same column is significant (P ≤ 0. 05).

Fig. 4.

Oxidative stress markers and antioxidant defense system of adult worms of H.contortus treated with oils. a Activation of catalase against ROS generated stress. Note a steady increase in CAT activity when exposed to a drug treatment with an increase of stress response synchronized with an increase of oils concentration. b Elevated glutathione transferase level due to drugs and oils stress. Note GPT showed an increased activity when exposure to increased concentrations of oils. c Cellular combat with ROS by increasing the reduced glutathione concentration. Note a significant increase in GSH content in drug and oils-treated helminths when exposure to increased concentrations. d Activity of NOS on treatment with oils. Note a subsequent decrease in NOS generation in the lowest concentration. There were a significant decrease in NO content in drug and oils-treated helminths. e Level of LPO when treated with oils. Note a significant decrease in LPO content in drug and oils-treated helminths. A subsequent decrease in LPO generation in the lowest concentration

Elevated glutathione transferase level

Conflicting to other antioxidant activities, GPT showed an increased activity when worms exposed to oils of increasing concentrations. In the control untreated groups, it was 337.27 µ/g tissue, while in albendazole-treated groups, it reached 891.26 µ/g tissue. For NBP 5.0%, NBP 2.5% and NBP 1.25%, it was 814.64 µ/g tissue, 730.42 µ/g tissue and 796.95 µ/g tissue, respectively. Meanwhile, for BP 5.0%, BP 2.5% and BP 1.25%, it was 795.70 µ/g tissue, 638.95 µ/g tissue and 635.15 µ/g tissue, respectively. Similarly, for NTT 5.0%, NTT 2.5% and NTT 1.25%, it was 822.52 µ/g tissue, 789.48 µ/g tissue and 722.52 µ/g tissue, respectively. For TT 5.0%, TT 2.5% and TT 1.25%, it was 817.62 µ/g tissue, 653.19 µ/g tissue and 518.65 µ/g tissue, respectively (Table 4 and Fig. 4).

Cellular combat with ROS

There was a significant increase in GSH content in drug and oils-treated helminths. The amount of GSH increased from 31.00 nmol/100 gm tissue in the control untreated groups to 91.38 nmol/100 gm tissue in albendazole-treated groups. Similarly, for NBP 5.0%, NBP 2.5% and NBP 1.25%, it was 96.73 nmol/100 gm tissue, 80 nmol/100 gm tissue and 74.95 nmol/100 gm tissue, respectively. Meanwhile, for BP 5.0%, BP 2.5% and BP 1.25%, it was 78.19 nmol/100 gm tissue, 77.64 nmol/100 gm tissue and 67.32 nmol/100 gm tissue, respectively. Similarly, for NTT 5.0%, NTT 2.5% and NTT 1.25%, it was 84.74 nmol/100 gm tissue, 79.61 80 nmol/100 gm tissue and 70.35 nmol/100 gm tissue, respectively. For TT 5.0%, TT 2.5% and TT 1.25%, it was 77.64 nmol/100 gm tissue, 72.41 nmol/100 gm tissue and 69.77 nmol/100 gm tissue, respectively (Table 4 and Fig. 4).

Determination of NOS on treatment with oils

NOS generation in response to oils treatment was monitored. The current findings exhibited a subsequent decrease in NOS generation in the lowest concentration. There were a significant decrease in NO content in drug and oils-treated helminths. The amount of NO decreased from 0.26 µ/mg tissue in the control untreated groups to 0.13 µ/mg tissue µ/mg tissue in albendazole-treated groups. Similarly, for NBP 5.0%, NBP 2.5% and NBP 1.25%, it was 0.18 µ/mg tissue, 0.15 µ/mg tissue and 0.15 µ/mg tissue, respectively. Meanwhile, for BP5%, BP2.5% and BP1.25%, it was 0.12 µ/mg tissue, 0.15 µ/mg tissue and 0.18 µ/mg tissue, respectively. Similarly, for NTT 5.0%, NTT 2.5% and NTT 1.25%, it was 0.17 µ/mg tissue, 0.13 µ/mg tissue and 0.19 µ/mg tissue, respectively. For TT 5.0%, TT 2.5% and TT 1.25%, it was 0.13 µ/mg tissue, 0.19 µ/mg tissue and 0.18 µ/mg tissue, respectively (Table 4 and Fig. 4).

Determination of LPO when treated with oils

The experimental results exhibited a subsequent decrease in LPO generation in the lowest concentration. There was a significant decrease in LPO content in drug and oils-treated helminths. The amount of LPO decreased from 2.40 nmol/100 mg tissue/hour in the control untreated groups to 0.82 nmol/100 mg tissue/hour in albendazole-treated groups. Similarly, for NBP 5.0%, NBP 2.5% and NBP 1.25%, it was 0.65 nmol/100 mg tissue/hour, 1.65 nmol/100 mg tissue/hour and 1.80 nmol/100 mg tissue/hour, respectively. Meanwhile, for BP 5.0%, BP 2.5% and BP 1.25%, it was 1.10 nmol/100 mg tissue/hour1.12 nmol/100 mg tissue/hour and 1.68 nmol/100 mg tissue/hour, respectively. Similarly, for NTT 5.0%, NTT 2.5% and NTT 1.25%, it was 0.63 nmol/100 mg tissue/hour, 1.23 nmol/100 mg tissue/hour and 1.91.nmol/100mg tissue/hour, respectively. For TT 5.0%, TT 2.5% and TT 1.25%, it was 1.04 nmol/100 mg tissue/hour, 1.03 nmol/100 mg tissue/hour and 0.89 nmol/100 mg tissue/hour, respectively (Table 4 and Fig. 4).

Scanning electron microscopy (SEM)

The untreated H.contortus anterior end showed a normal transverse striated cuticle and longitudinal ridges with a normal cuticle (Fig. 5a, b) and the vulvar flap of the posterior end female was thumb-like, adapted with longitudinal furrows, but sometimes showed polymorphism with a transverse striated surface (Fig. 5c). In albendazole-treated worms, the anterior end elucidated normal cuticular ridges (Fig. 5d). There were no furrowing or pores (Fig. 5e) and the vulvar flap showed shrinkage in the cuticle (Fig. 5f). Meanwhile, in NBP 5%-treated worms, various alterations consisted of damaged anterior end (Fig. 5g), the occurrence of pores and furrowing cuticle, pores in the intercuticular area, deep cuts in some areas of the cuticle (Fig. 5h), sloughed cuticle together with cuts in the vulvar flap region were found (Fig. 5i). Similarly, in NTT 5.0%-treated worms, sloughed cuticle, unclear cuticular ridges with pores (Fig. 5j, k) and small cuts in the cuticle of the vulvar flap region were observed (Fig. 5l).

Fig. 5.

Scanning electron microscopy of adult H.contortus worms post oil treatment. a Anterior end of adult H.contortus with a normal cuticle and clear cuticular ridges. b Transverse ridges of adult H.contortus with an intact cuticle and no pores or cut. c Normal vulvar flab. Note a thumb-like flab, adapted with longitudinal furrows with transverse striated surface without cuts. d Anterior end of albendazole-treated adult H.contortus with normal cuticular ridges. e Transverse ridges of albendazole-treated H.contortus cuticle. Note only some shrinkage of the cuticle. f Vulvar flab of albendazole-treated H.contortus. Note a shrunken flab. g Anterior end of NBPO-treated H.contortus. Note a damaged anterior end. h Cuticle of NBPO-treated H.contortus. Note the occurrence of pores and deep cuts in the cuticle and furrowing cuticle. i Vulvar flab of NBPO-treated H.contortus. Note a sloughed cuticle and cuts. j Anterior end of NTTO-treated H.contortus with unclear cuticular ridges. k Cuticle of NTTO-treated H.contortus. Note the occurrence of cuticular pores and small cuts. l Vulvar flab of NTTO-treated H.contortus. Note a sloughed cuticle

Discussion

Haemonchus contortus infection is an imperative disease infecting ruminants often in nations where sanitation and the economy are both weak. Previous literature revealed the resistance of Haemonchus contortus against various anthelmintic drugs. Cawthorne et al. (1983) revealed the resistance to benzimidazole's suppression of polymerizations by microtubules. Sangster et al. (1979) showed that the resistance to imidazothiazole is developing in tropical regions. The search for alternate sources to reduce parasite infection and enhance public health is prompted by the emergence of anthelmintic resistance and the existence of medication residues in the food chain. Antiphrastic agents extracted from plants have also been used inhuman and animal populations with limited scientific evaluation (Masood et al. 2013). It is known that H. contortus is resistant to the majority of commercially available anthelmintic medication classes, including imidazothiazoles, benzimidazoles, and macrocyclic lactones (Chagas et al. 2013). Drudge et al. (1964) denoted the first of chemoresistance of H.contortus against benzimidazole. Because essential oils contain components that have antiparasitic activity, using them is a superior option to the phytotherapeutic approach (Anthony et al. 2005). The substantial therapeutic effectiveness of M. alternifolia (tea tree) essential oil against ectoparasites and protozoa has already been demonstrated. (Baldissera et al. 2014; Pazinato et al. 2014), however, studies on the action of endoparasites are still insufficient (Payne et al. 2013). The genus Melaleuca, which includes M. artroviridis, M. stereophloia, and M. uncinata, was shown to exhibit noticeable activity when extracts of native Australian plants were evaluated for their effects on the development of horse cyathostominae larvae.

The current study conducted an anthelmintic effect of TTO against Haemonchus contortus. It has been found that albendazole had a high efficacy. Albendazole-treated adult Haemonchus contortus had been died after 4.5 h. Meanwhile, the egg reduction of albendazole-treated worms was 94.7%. The egg development and hatching of eggs were significantly reduced. Albendazole-treated groups showed a significant decrease in the hatched eggs and increase of the undeveloped eggs compared to untreated groups. This finding disagreed with Shahardar et al. (2014), Westers et al. (2016), Baihaqi et al. (2019), Hassanet al. (2021) who observed that greater anthelmintic resistance in small ruminants resulted in reduced efficacy for albendazole, oxfendazole, and ivermectin. The increased frequency of deworming and the low dose of anthelmintics contributed to the higher anthelmintic resistance (Sissay et al. 2006). This discrepancy might be attributed to the variation of the dosage as well as potency and manufacturing of the drug used. The current study revealed that TTO had an ant parasitic activity against Haemonchus contortus adult worm, eggs and larvae. Similarly, The TTO essential oil caused death of adult Haemonchus contortus after 3 h. Furthermore, NTTO essential oil induced death of adult worms 3 h post application. TTO and NTT induced a reduction in the oviposition. Meanwhile, the egg development and hatching of eggs were significantly reduced. After the exposure of eggs to various treatments, the development in the control untreated was 80%. Meanwhile, in all treated groups, the development were recorded zero (no L3 developed). Grando et al. (2016) showed that TTO and terp-4-ol were only slightly effective against H. contortus larvae and extremely effective against eggs. At a dosage of 3.5 mg/ml, the inhibition of hatching was 100%, and for TTO and terp-4-ol, the LC50 values were 0.43 and 0.63 mg/ml, respectively.

The approach that TTO is used to treat helminth and protozoan diseases is associated with its ingredients and its primary monoterpene. The antiparasitic properties of TTO, particularly its monoterpene extract 1,8-cineole, may be attributed to its ability to competitively block the enzyme acetylcholinesterase (AchE) in parasites (Mills et al. 2004). It has been indicated that in certain parasitic nematodes, inhibiting AchE activity might lead to increased levels of acetylcholine (Ach) and excessive activation of nicotinic acetylcholine receptors (nAchR)(Wolstenholme 2011). Through membrane depolarization and an influx of Na + , K + , or Ca2 + , a high level of Ach produces muscle contraction and spastic paralysis in some worms. The World Association for the Advancement of Veterinary Parasitology (WAAVP) has recommended that potential anthelmintics be classified as highly effective if their efficacy is greater than 98%, effective if their efficacy is between 90 and 98%, moderately effective if their efficacy is between 80 and 89%, and ineffective if it is less than 80% (Wood et al. 1995).

Eucalyptus citriodora and Eucalyptus globulus essential oils also caused a decrease in egg hatching (98.8% and 99.3%, respectively) in vitro (Macedo et al. 2009, 2011). Egg count was dropped by 76.57% using Eucalyptus staigeriana essential oil (Macedo et al. 2010) in vivo. Katiki et al. (2011) assessed the essential oils of Cymbopogon schoenanthus, Cymbopogon martinii, and Mentha piperita against trichostrongylidae parasites using the EHT and discovered that the LC 50 values were just 0.04 mg/ml, 0.13 mg/ml, and 0.26 mg/ml.

It has been demonstrated that the peppermint essential oil precious against the protozoan parasite, Giardia (Vidal et al. 2007). Other essential oils or their constituents might also possess anticoccidial or antiminematic properties (Benchaar et al. 2008). Hassan et al. (2021) reported that CEEs of A. herba-alba and B. aegyptiaca had a substantial in vivo anthelmintic effect compared to albendazole on lambs experimentally infected with H.contortus. The group treated with B. aegyptiaca CEE was first, followed by the group treated with A. herba-alba CEE. After three days PT, they observed a considerable decrease in FECs, which persisted for the duration of the experiment. For the CEE of B. aegyptiaca and A. herba-alba, they likewise performed an excellent FECR (100% and 85.1%) from the seventh day of treatment until the conclusion. Also, Idris et al. (1982) revealed by A. herba-alba's beneficial effects on a goat with haemonchosis. Zhu et al. (2013) used the essential oil of Artemisia lancea and its major constituents were 1,8-cinelone and camphor. The ovicidal activity of A. lancea and 1, 8-cinelone oil at a dose of 10 mg/ml was 99.4% and 74.8%, respectively, with LC50 values of 1.82 and 4.64 mg/ml. For instance, limonene can be found in the oils of several anthelmintic plants, including Eucalyptus staigeriana, Mentha piperita, Lippia sidoides, and Cymbopogon martini (Katiki et al. 2011; Carvalho et al. 2012; Ribeiro et al. 2014). This substance, which at most tested concentrations, completely inhibits H. contortus eggs and larvae in vitro, makes up 96.0% of Citrus sinensis essential oil composition (Gaínza et al. 2015). Also, linalool acetate is known for its anthelmintic activity against Ascaris lumbricoides (Kaplan et al. 2012).

The harmful effects of TTO and BPO on infectious helminths are countered by oxidative stress-responsive components in H.contortus. Owing to the production of stress-related oxide synthase, helminths combat circumstances by changing their metabolic processes. The harmful impact of oils on the helminth's body interacts with its molecules as part of regular metabolic processes, promoting the creation of free radicals as a defense strategy against oxidative damage and boosting certain enzyme activities. Like CAT, GST and GSH and consequently decreasing LPO and NOS levels.

The current study conducted an increased activity of antioxidant enzymes (CAT, GST and GSH) when exposed to different oils of ascending concentrations due to increasing the stress. Meanwhile, there was a decreased activity of oxidative enzymes (LPO and NOS) when exposed to various oils of descending concentrations. This agreed with Goel et al. (2020) who stated that when adult worms treated with Cuminaldehyde (CA) for 3 h, there was a generation of oxidative stress resulted in toxic effect of CA on the body of the helminth which promotes the creation of free radicals brought on by oxidative damage defense mechanisms and interacts with cellular molecules as part of regular metabolic processes, leading to an elevation in enzymatic factors of CAT, GPx and GSH.

For the first time in Egypt, the current study conducted the potential inhibitory effect of Black pepper (Piper nigrum L.) essential oil against eggs, larvae and adult stages of H. contortus, one of the most economically important parasitic nematode with total costs resulted from losses of 120 billion of USD annually worldwide (DeVaney et al. 1992; Waruiru et al. 1997; Kaplan 2004; Schwab et al. 2005; Entrocasso et al. 2008).

In the present work, BPO 5.0% caused death of adult Haemonchus contortus after 2.5 h of application while NBPO 5.0% caused death 1.5 h post application. Concerning the egg reduction, BPO 5.0% and NBPO 5.0% induced a significant reduction in the oviposition to 93.8% and 97.9%. The egg development and hatching were significantly (P ≤ 0.05) reduced. In BP 5.0%-treated eggs, they were 8.00 ± 0.37, 18.00 ± 1.59 and 74.00 ± 1.26 for hatched, developed not hatched and undeveloped, respectively. Similarly, for NBP 5.0%, they were 2.33 ± 0.56, 9.33 ± 0.21 and 88.33 ± 0.56, respectively. Gaínza et al. (2016) observed that P. aduncum L. exhibited anthelminthic effects against H. contortus and Trichostrongylus colubriformis larvae. The LDT yielded IC50 values of 0.54 and 0.64 μg/mL, respectively. Adult H. contortus likewise completely stopped moving when exposed to that component at 20 μg/mL. Moreover, the use of EHT exhibited that P. aduncum LEO was 95% effective against H. contortus eggs at a dose of 12 mg/mL (Oliveira et al. 2014).

Scanning electron microscopy (SEM)

The current study revealed that untreated H.contortus anterior end showed a normal transverse striated cuticle and longitudinal ridges, with normal cuticle and the vulvar flap of the posterior end female was thumb-like, adapted with longitudinal furrows, but sometimes showed polymorphism with a transverse striated surface which agreed with Baihaqi et al. (2020). Tresia et al. (2016) stated that anthelmintic activity carried out by active compounds of plants damaged the cuticle and changed the shape of the pore and permeability of the worm cuticle. Sambodo et al. (2018) added that the cuticular alterations occurred by interactions with the Biophytum petersianum aqueous extract, which is vital for nutrient absorption and motility. The cuticle gives the shape of the worm. In the host's digestive tract, it also plays a role in the motility and interactions with the parasite environment, including metabolic interactions (Fetterer and Rhoads 1993; Page 2001; Page and Winter 2003). Related functional abnormalities as well as structural cuticular alterations were place, which may have contributed to potential limitations in the nematodes' ability to move freely. In albendazole-treated worms, the anterior end showed normal cuticular ridges. There were no furrowing or pores and the vulvar flap showed shrinkage in the cuticle. Meanwhile, in NBP 5.0%-treated worms, there were damaged anterior end, pores and furrowing cuticle, cuts, pores in intercuticular area, deep cuts in some areas of the cuticle as well as sloughed cuticle and cuts in the vulvar flap region. Similarly, in NTT5%-treated worms, there were sloughed cuticle, unclear cuticular ridges and pores and small cuts in the cuticle in the vulvar flap region. Currently, we found that the nanoemulsion increased the antiparasitic activity of both TTO (decreased time of death of adult worm from 2.5 h to one hour and increase the reduction of eggs from 81 to 95.4% and increased the degeneration of egg and L1 also it increased the effect of the oil on hatchability of treated eggs) and in BPO and also their improve the effect (decreased time of death of adult worm from 2 h to one hour and increase the reduction of eggs from 93.8 to 97.9% and increased the degeneration of egg and L1 also it increased the effect of the oil on hatchability of treated eggs). NBPO had much more effect than NTTO BPO is cheaper than TTO, so BPO is more recommended against H.contortus in infected sheep.

The current study concluded that there is an anthelmintic potency of the BPO and TTO and their nanoemulsion forms against H. contortus much higher than widely used anthelminthic, albendazole.

Author contributions

HIM; samples collection, lab work. WMA; designing, executing and drafting the manuscript, OMA; data analysis and statistical analysis. KhME; revising, photo editing. All the authors have given their consent to submit the manuscript in the Journal.

Funding

No funding sources.

Data availability

The datasets uses and/or analyzed in the manuscript are available from the corresponding author upon reasonable request.

Code availability

Not applicable.

Declarations

Conflict of interest

No conflict of interest in the submission of this manuscript, and manuscript is approved by all authors for publication.

Ethics approval

The authors assert that all procedures contributing to this work comply with the ethical standards of the institutional Animal Care and Use Committee Beni-Suef University (BSU-IACUC 022-419).