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Introduction
Gastrointestinal nematodes are a large concern in agriculture, because they substantially reduce animal productivity and cause economic losses for breeders. The cost of helminth infections in ruminants in 18 countries (including 13 European countries) has been estimated at 1.8 billion €, where 81 % of the total cost was assigned to production losses and 19 % was assigned to treatment costs (Charlier et al., 2020). The interest in studying the strategies for controlling nematode infections is thus constantly growing. The use of synthetic anthelmintics is still the preferred strategy, but it carries a risk of the development of anthelmintic resistance (Stear et al., 2007). Synthetic anthelmintics also pose the risk of the presence of their residues in animal products for consumers and the environment (Jedziniak et al., 2009; Biswas et al., 2012). The blood-sucking nematode Haemonchus contortus, whose presence causes severe anemia and the loss of proteins, is the most harmful nematode in small ruminants and is most frequently connected with anthelmintic resistance (Besier et al., 2016). The objective of most methods for controlling parasites is not to totally eliminate the parasites from the herd, but to maintain population at levels that will not affect the health of the host population (Selemon, 2018). Nematodes can be controlled by alternative ways, such as the use of nematophagous fungi, vaccination, rotational grazing or adjustment of feed rations (Molento et al., 2013).
The use of plants in the treatment of human and animal diseases dates back to the beginnings of our species. In the first half of the 20th century, plants worldwide formed the basis of drugs in both human and veterinary medicine (Hammond et al., 1997). Willcox et al. (2001) estimated that more than 30 % of drugs placed on the market are directly or indirectly of natural origin. Plants or plant components have been used for dehelminthization for several centuries (Waller et al., 2001). More comprehensive research has been conducted over the last 10 – 15 years to evaluate the in vitro and in vivo activities of bioactive plants under experimental and field conditions (Sandoval-Castro et al., 2012). Parallel in vivo and in vitro studies are recommended for finding promising herbal alternatives to synthetic antiparasitic drugs (Váradyová et al. 2018). Several studies have already confirmed the antiparasitic properties of plants and their secondary plant metabolites tannins or flavonoids (Spiegler et al., 2017). The most important antiparasitic effect has been attributed to forage legumes containing condensed tannins, such as sainfoin (Heckendorn et al., 2006; Komáromyová et al., 2022), birdsfoot trefoil (Marley et al., 2003), sulla (Tzamaloukas et al., 2006) or sericea lespedeza (Dykes et al., 2019). Extracts from various medicinal plants, however, also have anthelmintic properties (Carvalho et al., 2012; Mravčáková et al., 2019; Pinto et al., 2019).
Changes in the ultrastructure of cuticles of parasites of small ruminants after exposure to tannin-rich materials were first described by Hoste et al. (2006). The effects of the consumption of individual plant species on the ultrastructural changes in nematodes have also been studied (Kommuru et al., 2015; Desrues et al., 2016; Martínez-Ortíz-de-Montellano et al., 2013), but no evidence of the effect of herbal mixtures of multiple plant species has been found. Data on ultrastructural changes caused by blends of medicinal plants under in vitro conditions is also lacking, but some studies have nonetheless focused on individual plant species and their compounds (Ribeiro et al., 2017; Adamu et al., 2019; Cavalcante et al., 2020).
The objective of our study was therefore to monitor the efficacy of a mixture of medicinal plants on superficial ultrastructural changes under in vivo and in vitro conditions and on the ovicidal activity of a herbal mixture against H. contortus. Individual plants contained in the herbal mixture (Herbmix) were chosen because of their previously described antiparasitic properties (Váradyová et al., 2018) and their potential availability on pasture in temperate climatic zones.
Material and Methods
In vivo analysis
Adult parasites were obtained from lambs in a complex study evaluating the antiparasitic effect of Herbmix; the complete methodology of this in vivo experiment was previously described (Komáromyová et al., 2021). Lambs of the Improved Valachian breed with the same age and similar body weight were supplemented with the “Herbmix” mixture of dry medicinal plants (100 g dry matter/d/animal) and an unsupplemented group was used as a control. After a period of acclimation, the lambs were orally infected on day 0 (D0) with approximately 5000 larvae of the MHCo1 strain of H. contortus susceptible to anthelmintics. The animals were re-infected on D49 and D77 with 3000 larvae of the same strain to simulate natural conditions on pasture. Lambs from the control group were fed hay containing no medicinal plants. Herbmix supplementation started on D0, continued throughout the experimental period and was terminated on D119 when the animals were slaughtered and necropsied. The lambs were euthanized 17 wk after infection following the rules of the European Commission (Council Regulation 1099/2009) for slaughtering procedures. Several dozen adult worms for observation by scanning electron microscopy (SEM) were randomly collected at necropsy from the abomasa of animals from the Herbmix and control groups.
Herbmix composition
The Herbmix contained nine medicinal plants: marshmallow (Althaea officinalis), butterbur (Petasites hybridus), elecampane (Inula helenium), ribwort plantain (Plantago lanceolata), rosemary (Rosmarinus officinalis), fennel (Foeniculum vulgare), goldenrod (Solidago virgaurea), fumitory (Fumaria officinalis) and hyssop (Hyssopus officinalis), complete composition of Herbmix was previously decribed by Komáromyová et al. (2021).
In vitro analyses
Egg hatch test
An egg hatch test was performed to determine the ovicidal activity of an aqueous extract prepared from ground Herbmix. The preparation of aqueous extracts has previously been described by Váradyová et al. (2018). H. contortus eggs used in the EHT were obtained from freshly rectally collected feces from experimentally infected lambs (control group). The methodologies for isolating eggs and for the EHT have previously been described by Váradyová et al. (2018). The final concentrations of Herbmix in the EHT obtained by serial dilution were 25, 12.5, 6.25, 3.125 and 1.563 mg/mL. We therefore calculated the median effective dose (ED50), which is the concentration that inhibits hatching in 50 % of eggs.
In vitro exposure to the Herbmix extract
The methodology for exposing adult worms to Herbmix extracts was conducted as described by Martínez-Ortíz-de-Montellano et al. (2013), with slight modifications. Adult worms used for the in vitro experiment were collected after slaughter from experimentally infected lambs from the control group (described in the section “In vivo analysis”) that were fed hay containing no medicinal plants. Adult worms were recovered from the abomasum, washed with a physiological saline solution (0.9 % NaCl) and placed into a Petri dish containing the same solution to remove abomasal content. All procedures were performed at 37 °C. Female worms were then separately placed into 24-well plates containing either the Herbmix extract (25 mg/mL) for the treated group (n=12) or phosphate buffered saline (PBS) for the control group (n=12). All worms were incubated for 24 h and then fixed for scanning electron microscopy.
Scanning electron microscopy
Randomly chosen adults of both sexes from those collected from both the treated and control lambs, and all specimens in the in vitro experiment, were rinsed in saline, fixed in a hot 4 % formaldehyde solution and stored in 70 % non-denatured ethanol. All worms were then dehydrated using an ascending ethanol series (80-9096-100 %; 15 min. at each concentration) and chemically dried in hexamethyldisilazane (Merck KGaA, Darmstadt, Germany). Dried specimens were transversely cut into three pieces. The anterior and posterior parts of their bodies were attached to aluminum SEM targets by double-sided tape, covered by gold in a JEOL JFC 1300 Auto fine coater and examined using a JEOL JSM 6510LA scanning electron microscope. The post-processing of the SEM photomicrographs included only the adjustment of brightness/contrast and blacking the background.
Ethical Approval and/or Informed Consent
This study was conducted following the guidelines of the Declaration of Helsinki and national legislation in the Slovak Republic (G.R. 377/2012; Law 39/2007) for the care and use of research animals. The experimental protocol was approved by the Ethical Committee of the Institute of Parasitology of the Slovak Academy of Sciences on 22 November 2020 (protocol code 2020/21).
Results
Scanning electron microscopic examination of H. contortus from the in vivo experiment
Scanning electron photomicrographs were acquired for six individuals (four females, two males) from each of the Herbmix and control groups. The ultrastructure of the body surface of the worms generally did not differ significantly between the groups. The cuticles were similarly smooth with straight, i.e. not sinuous, physiological longitudinal ridges (Fig. 1 H1, C1) throughout their bodies. The only observable differences were small protuberances around the bases of the cervical papillae and weak ridges in the vulvar area of the worms from the lambs fed the Herbmix-supplemented diet (Fig. 1 H2, H3), but which were also detected to some extent in the control group (Fig. 1 C2, C3). The surface ultrastructure of the copulatory bursae of males did not differ between the groups in a way, which could be attributed to an effect of the Herbmix. The cuticles of all worms, however, had many unnatural folds (e.g. Fig.1 C1, H4), which were considered to be artifacts of processing for SEM, because they occurred in both groups.
Fig. 1
Scanning electron photomicrographs of Haemonchus contortus from the in vivo experimental groups. H1–H4 depict specimens collected from lambs fed a diet enriched with a Herbmix extract, and C1–C4 depict specimens from the control animals. H1 and C1, cephalic end of the body, both females; H2 and C2, region near the cervical papillae, both females; H3 and C3, vulvar region of the body, both females; H4 and C4, posterior end with the copulatory bursa, both males. Note the cephalic end of the female body (C1) and the posterior part of the male body (H4), with several artificial folds (asterisk), small protuberances (arrowhead) in the region anterior to the base of a cervical papilla (H2) and weak ridges (arrow) on the cuticle in the vulvar region (H3).
Egg Hatch Test
The effects of Herbmix in the EHT are presented in Table 1. Hatching was inhibited by 83 % at the highest concentration (25 mg/mL) of the Herbmix extract. Each concentration of Herbmix extract tested significantly negatively affected hatching compared to the control (p<0.001). The inhibition of hatching was dose-dependent (Fig. 2). ED50 and ED99 were 6.517 and 315.294 mg/mL, respectively.
Fig. 2
Dose-response relationship of the Herbmix aqueous extract against Haemonchus contortus in the EHT.
Hatching and ovicidal activity at the concentrations of the Herbmix aqueous extract tested in the EHT.
Herbmix concentration (mg/mL)
Ovicidal effect (%)
Hatching (%)
p value
25
83
17
< 0.001
12.5
71
29
< 0.001
6.25
45
54
< 0.001
3.125
34
66
< 0.001
1.563
15
85
< 0.001
Control
1
99
-
EHT – Egg Hatch Test
Scanning electron microscopic examination of H. contortus from the in vitro experiment
Three worms fixed in the in vitro experiment were randomly chosen from each of the Herbmix and control (PBS) groups. SEM did not identify any obvious damage, which the Herbmix decoction could cause on their superficial structures. The cuticle at the cephalic ends of the worms in both groups was smooth, with the lack of any ruptures (Fig. 3 H1, C1). The physiological longitudinal ridges were slightly sinuous, and small protuberances occurred around the bases of the cervical papillae, but these features were observed in both the treated and control groups (Fig. 3 H2, C2). The rough cuticle covered the vulvar flaps and lateral buttons of the worms from both groups, but it seems to be physiological appearance of these structures because of its presence also in control group, and weak transverse wrinkles were observed anterior and posterior to the vulvar flaps (Fig. 3 H3, C3). Similar to the in vivo group, all specimens, i.e. from both the treated and control groups, had deformed body surfaces, which were attributed to the preparation of the SEM samples (see artifacts in Fig. 3 C1, H2, H3, C3)
Fig. 3
Scanning electron photomicrographs of Haemonchus contortus from the in vitro experimental groups. H1–H3 depict specimens treated with the Herbmix extract diluted in PBS, and C1–C3 depict specimens from the control group kept in PBS. H1 and C1, cephalic end of the body; H2 and C2, region around a cervical papilla; H3 and C3, vulvar region with a lateral button. Note the slightly sinuous physiological longitudinal ridges (arrows), weak transverse wrinkles (arrowheads) and artifacts of sample preparation (asterisks).
Discussion
The demand for organic animal production and for minimizing the levels of chemicals in animal products is growing with the popularization of ecofriendly lifestyles. The presence of parasites is usually an inherent part of intensive livestock production, and breeders and scientists are looking for ways to minimize the damage caused by parasitic infections. The available literature indicates that 458 studies have been published and 187 plant species have been monitored for their antiparasitic effects against H. contortus over the last 30 years (Ali et al., 2021). Local and exotic plants in countries around the world are being explored for their activity against parasitic organisms under in vivo and/or in vitro conditions. Our studies are participating in the research for finding promising natural alternatives to synthetic anthelmintics. After the promising results obtained in our previous studies with herbal mixtures (Váradyová et al., 2018b; Komáromyová et al., 2021), we are focusing on possible ultrastructural changes to the adult stage of H. contortus. To our knowledge, this study is the first to examine the effects of herbal mixtures on ultrastructural changes in H. contortus.
The content of phytochemicals in plants and their extracts is the most important factor responsible for anthelmintic effects. Plant secondary metabolites have been discussed and contemplated as tools for the control of gastrointestinal nematodes in small ruminants (Hoste et al., 2016). Herbmix has tested positive for tannins, saponins, alkaloids, terpenoids, flavonoids and steroids (Komáromyová et al., 2021). The mechanism of action of condensed tannins against parasites, though, remains unexplained. What is known is that the surface structures (cuticle) of nematodes contain collagen proteins (Thompson & Geary, 1995). Tannin-binding proteins have been identified in the saliva of deer but not the saliva of cattle or sheep (Austin et al., 1989), so tannins may be able to pass into the stomach or intestines unbound and interact there with parasites and their structures. Tannins can bind proteins or amino acids and modify their chemical properties to form soluble complexes or precipitates, which can affect protein function or inhibit enzymatic activity (Rinaldi & Moio, 2020). Tannins have been hypothesized to bind to cuticular proteins and cause ultrastructural changes that can be seen by scanning electron microscopy (Hoste et al., 2006).
In Figure 1 worms differed little between the Herbmix and control groups. Some differences were observed, but these were insufficient to confirm the effect of Herbmix on the superficial structures of H. contortus. Significant antiparasitic effects of Herbmix on fecal egg counts, live-weight gains and abomasal worm counts in vivo, however, have been reported (Komáromyová et al., 2021). These results led us to hypothesize that antiparasitic effects could be achieved by improving the health of the host or by impairing internal structures of nematodes more than by directly harming the surface structures of adult parasites. Substances harmful to superficial structures may also not have been present. The concentrations of active plant substances in lambs after metabolic processes during digestion may also have been too low. Another study investigating the effects on structures of H. contortus in goats supplemented with tannin-rich sericea lespedeza, however, confirmed damage to the cuticular surface in a treated group (Kommuru et al., 2015). H. contortus isolated from goats fed with tzalam or sainfoin also had signs of surface damage, e.g. ridges on the cuticle and aggregates around the buccal capsule, vulva and anus (Martínez-Ortíz-de-Montellano et al., 2013). A similar study performed with sainfoin-fed calves infected with Ostertagia ostertagi and Cooperia oncophora found local cuticular damage (Desrues et al., 2016). Similar studies with sheep have not yet been conducted.
In vitro tests are commonly used due to their low cost, quick outcome and possibility of testing purified compounds without interfering with metabolites or components present during digestion. Reproducing the same experiments in vivo, however, is difficult, mostly due to differences in dosage and absorption. Substances that are effective under in vitro conditions do not always have same effect under in vivo conditions due to differences in metabolism and bioactivity in the host organism (Ferreira et al., 2013). A comparison of methanolic and aqueous extracts of the same plants confirmed differences in ovicidal activity in one plant (Váradyová et al., 2018). In vitro tests are also usually performed with eggs or free-living larval stages rather than adults because of their viability outside their host (Athanasiadou & Kyriazakis, 2004). All these factors led us to conduct in vivo and in vitro tests and to include adult nematodes for assessing the efficacy of Herbmix.
One hypothesis of the possible mechanism of action of tannins is that they could have antiparasitic properties and could directly affect the biology of parasites (Hoste et al., 2006). This hypothesis has been substantiated by many in vitro studies (Yoshihara et al., 2014; Castañeda-Ramírez et al., 2018; Silva Soares et al., 2018) where direct effects of tannins were assumed. The ovicidal effects of the individual plants in Herbmix were previously examined, with the strongest ovicidal activity observed for F. officinalis and A. officinalis (Váradyová et al., 2018a). The ovicidal effect of Herbmix in our study was obvious, but with a high ED99, the ability to achieve the required concentration and therefore antiparasitic effect in vivo is uncertain.
In vitro studies with SEM and the effects of plants on adult nematodes always have complicated processes or chemical solvents behind the extraction of active plant substances (Martínez-Ortíz-de-Montellano et al., 2013; Andre et al., 2016; Adamu et al., 2019; Cavalcante et al., 2020). One study (Ribeiro et al., 2017) used an aqueous extract of Spigelia anthelmia and a relatively simple process of decoction. This study found ultrastructural changes to the cuticle in the cephalic region, unlike in our study, even though the methods of extraction were very similar. The difference between findings may have been caused by the diverse plants examined. The accuracy of the method of extraction has been widely discussed, because plants cannot be similarly processed by the digestive tracts of animals (Mueller-Harvey et al., 2018). Alternative therapies avoid the use of synthetic substances, so results confirming damaging effects on parasites in vitro are not adequate for dehelminthization in the ruminant husbandry. Aqueous extracts were also used in a study of Acacia mearnsii and H. contortus, where cuticular ruptures and wrinkling were observed, but the stock extract was commercially prepared (Yoshihara et al., 2015). Structural cuticular changes that could inhibit the motility of worms were described in a study using aqueous extracts of bamboo leaves, although the method of extract preparation was not described (Widiarso et al., 2021).
Resilience of parasites could be also influenced by their age. Ultrastructural changes to the cuticle and in the vulvar region after in vitro exposure to carvacrol and its derivates originating from essential oils of Lamiaceae species have been observed (Andre et al., 2016). These authors used adult H. contortus collected 4 wk after the infection of lambs. Santos et al. (2014) established the prepatent period of H. contortus at between 18 and 22 d, but egg output peaked on D52 post-infection. These data suggest that the worms could be more resilient to external influences 7 wk after infection. Younger worms may generally be more sensitive to ultrastructural damage and external factors compared to our worms isolated 17 wk after infection, disregarding the type of plant extract examined. Exposure of adult H. contortus to an acetone extract of agrimol G (isolated from leaves of Leucosidea sericeza) for 3 h caused distinct surface disruptions (Adamu et al., 2019). The worms in our study, however, did not have any signs of breakage, even though exposure time was 8-fold longer.
Conclusion
Changes in the surface structures of adult H. contortus after in vivo and in vitro contact with a herbal mixture were not observed under a scanning electron microscope. Aqueous extracts of Herbmix significantly inhibited hatching even at the lowest concentration. Together with our previous findings, this study suggests that Herbmix may be effective during the early developmental stages of H. contortus by directly damaging the internal structures of adult worms or by indirectly modulating immunity.
