Effects of herbal nutraceuticals and/or zinc against Haemonchus contortus in lambs experimentally infected

Background The gastrointestinal parasitic nematode Haemonchus contortus is a pathogenic organism resistant to several anthelmintics. This study assessed the efficacy of a medicinal herbal mixture (Herbmix) and organic zinc, as an essential trace element for the proper functioning of both unspecific and specific immune defensive mechanisms, against experimental infections with H. contortus in lambs. All lambs were infected orally with approximately 5000 third-stage larvae of a strain of H. contortus susceptible to anthelmintics (MHco1). Twenty-four female lambs 3–4 months of age were divided into four groups: unsupplemented animals (control), animals supplemented with Herbmix (Hmix), animals supplemented with organic zinc (Zn) and animals supplemented with Herbmix and organic zinc (Hmix+Zn). Eggs per gram (EPG) of faeces were quantified 20, 28, 35, 42, 49, 56, 62 and 70 d post-infection and mean abomasal worm counts were assessed 70 d post-infection. Samples of blood were collected from each animal 7, 35, 49 and 70 d post-infection. Results Quantitative analyses of the bioactive compounds in Herbmix identified three main groups: flavonoids (9964.7 μg/g), diterpenes (4886.1 μg/g) and phenolic acids (3549.2 μg/g). Egg counts in the lambs treated with Hmix, Zn and Hmix+Zn decreased after 49 d. The EPGs in the Zn and Hmix+Zn groups were significantly lower on day 56 (P < 0.05 and P < 0.01, respectively), and the EPGs and mean worm counts were significantly lower on day 70 in all supplemented groups (P < 0.05 and P < 0.01). Hemograms of complete red blood cells of each animal identified clinical signs of haemonchosis after day 35. Serum calprotectin concentrations and IgA levels were significantly affected by treatment. The treatment influenced serum malondialdehyde concentrations (P < 0.05) and sulfhydryl groups (P < 0.01) of antioxidant status. The mineral status was unaltered in all lambs. Conclusion A direct anthelmintic impact on the viability of nematodes was not fully demonstrated, but the treatments with herbal nutraceuticals and zinc likely indirectly contributed to the increase in the resistance of the lambs to nematode infection.


Background
Plant secondary metabolites (PSMs) of medicinal herbs, which have been used for centuries in traditional medicine and veterinary practice to treat various digestive or parasitic disorders, have important biological activities [1][2][3]. PSMs are bioactive compounds with applications in nutraceuticals and functional food. Compounds with nutraceutical activity [4,5] however, are mostly produced chemically, but new demands for a sustainable life are gradually favoring natural compounds, mainly those derived from herbs [6]. A recent study [7] has summarized information from 2001 to 2016 on polyphenols demonstrating promising anthelmintic activities of polyphenols that can be used as an additional or alternative treatment to current standard anthelmintics and as a base for the development of new substances that are urgently needed in human and veterinary medicine.
Chemoprophylaxis against the gastrointestinal parasitic nematode Haemonchus contortus by the repeated application of anthelmintics increases the risk of residues in food products and the development of anthelmintic resistance. Studies of foods containing healthy additives and having medicinal benefits in parasitized ruminants indicate that herbs with bioactive compounds represent a promising option for reducing nematode infections in small ruminants and for applications under farm conditions [8][9][10]. Phytotherapeutic treatments in traditional medicine [11] combined with the pharmacology of PSMs [12] and self-medication of ruminants against gastrointestinal parasites [13] can be used as an alternative strategy to control gastrointestinal helminths of small ruminants. The ability of the host to resist infection by gastrointestinal nematodes, though, is dependent on the development of a protective acquired immune response [14]. Nematode infections may also induce the production of reactive oxygen species, which may damage the parasites but generate oxidative stress in the hosts [15]. The trace element zinc is essential for the proper functioning of both unspecific and specific immune defensive mechanisms and reduces the impact of many diseases by preventing disassembly of the immune system [16,17]. Zinc is essential for the improvement of the immune functions because the efficiency of immunological responses, mainly intestinal immunity, against gastrointestinal nematodes depends on the zinc status and Zn nutrition [17]. The type of diet and availability of minerals are directly associated with the susceptibility of animals to parasites. Targeted nutrition and in vitro protocols for the mass production of PSMs have received much attention, but little emphasis has been placed on nutraceutical activity and analysis [6]. Supplementing diets with both medicinal herbs and zinc has therefore been hypothesized to affect the life cycle of H. contortus, have a direct anthelmintic impact on the viability of nematodes and may provide a better defense against oxidative stress of lambs, with an indirect impact on an increase in the resistance of hosts to parasitic infections of H. contortus. In this study the mix of traditional medicinal herbs (Herbmix) consist of herbs typical for Central Europe. These herbs were chosen based on information about their phytotherapeutic properties from traditional ethnomedicine practice.
Our goals were to (1) identify the main bioactive compounds of a medicinal herbal mixture (Herbmix) and (2) determine the effect of dietary supplementation with Herbmix and organic zinc (Zn-glycinate) on mean live-weight gain, parasitological status, hematological parameters, inflammatory response, antioxidative status and mineral status of lambs experimentally infected with H. contortus.

Animals, diets and experimental design
Twenty-four female lambs (Improved Valachian) 3-4 months of age with initial body weights of 15.12 ± 1.58 kg were housed in common stalls for 7 d for acclimatization to feeding, with free access to water. Each animal was fed a concentrate (500 g dry matter (DM)/d), Herbmix (a non-commercial product, 100 g DM/d) and meadow hay (ad libitum). The concentrate was composed of 37% wheat bran, 20% soybean meal, 23% rolled oats and 20% maize meal. After this adaptation period, all parasite free lambs were separated into four distinct pens, infected orally with approximately 5000 third-stage (L3) larvae of a strain of H. contortus susceptible to anthelmintics MHco1 (MOSI) which is susceptible to all the main classes of anthelmintics. It has been maintained since the late 1950s, and is thought to have been isolated in East Africa. Animals were randomly divided based on their live-weight into four groups of six animals each (n = 6/group, one stall per group): unsupplemented animals (control, C), animals supplemented with Herbmix (Hmix), animals supplemented with a zinc chelate of glycine hydrate -Glycinoplex-Zn 26% (Zn) and animals supplemented with both Herbmix and zinc (Hmix+Zn). The number of animals used in experiment was assigned according to VICH GL13 guidelines. The experimental period lasted 70 days (during summer) and the animals were housed in sheep farm. Herbmix is a mixture of dry herbs obtained from commercial sources (AGROKARPATY, Plavnica, Slovak Republic and BYLINY Mikeš s.r.o., Číčenice, Czech Republic): roots of marshmallow (Althaea officinalis L.), butterbur (Petasites hybridus L.) and elecampane (Inula helenium L.); leaves of ribwort plantain (Plantago lanceolata L.) and rosemary (Rosmarinus officinalis L.); seeds of fennel (Foeniculum vulgare Mill.) and stems of goldenrod (Solidago virgaurea L.), fumitory (Fumaria officinalis L.) and hyssop (Hyssopus officinalis L.). The Herbmix contained 11.8% each of A. officinalis, P. hybridus, I. helenium, P. lanceolata, R. officinalis, S. virgaurea, F. officinalis and H. officinalis and 5.6% F. vulgare. The Herbmix was stable throughout the experiment, mixed daily with the commercial concentrate from day D7 to D70. Aliquots of the zinc supplement were directly mixed with the concentrate for each feeding to provide an additional 60 mg zinc/kg concentrate. The allowed upper limits of zinc in complete feed is 120 mg/kg for food-producing animals [18]. Herbal and zinc supplementation began on D7. The chemical compositions of Herbmix, meadow hay, commercial concentrate and concentrate + Glycinoplex-Zn are given in Table 1. The lambs' wool was clipped on D3. The lambs were weighed on D7, D35, D49 and D70. Faeces were collected from the rectum, and the number of eggs per gram (EPG) of faeces was quantified on D0, D20, D28, D35, D42, D49, D56, D62 and D70. Samples of blood were collected from each animal on D7, D35, D49 and D70.

Chemical measurement and analysis
The dietary substrates (Herbmix, meadow hay, concentrate and concentrate + Zn-glycinate) were analyzed in triplicate for DM (No. 967.03), ash (method no. 942.05) nitrogen (method no. 968.06) and crude protein (method no. 990.03) using standard methods as described by AOAC [19]. The acidic-detergent fiber and neutraldetergent fiber contents were analyzed as described by Van Soest et al. [20] using an ANKOM 2000 fiber analyzer (ANKOM Technology, Macedon, USA) with heat-stable α-amylase.

Analysis of phenolic acids, flavonoids and diterpenes
The plant samples were ground to a fine powder, and 100 mg were extracted three times with a Dionex ASE (Accelerated Solvent Extractor, ThermoFisher, Sunnyvale, CA, USA) in 80% MeOH for 15 min (three static cycles, 5 min each), at 1500 psi solvent pressure, 100°C cell temp., flush 150%. The extracts were evaporated to dryness, dissolved in 1 mL of Milli-Q water (acidified with 0.2% formic acid) and purified by Solid Phase Extraction (SPE) using C18 Sep-Pak cartridges (1 cm 3 , 360 mg, Waters Corp., Milford, MA). The cartridges were washed with 0.5% methanol to remove carbohydrates, and then washed with 80% methanol to elute phenolics. The phenolic fraction was evaporated and dissolved in 1 mL of 80% methanol (acidified with 0.2% formic acid). The sample (intense yellow color without chlorophyll) was than centrifuged (18,766×g, 5 min) before spectrometric analysis. All analyses were performed in triplicate for three independent samples and stored in a freezer at − 20°C before analysis.

Ultra-high-resolution mass spectrometry (UHRMS)
The Herbmix bioactive compounds were analyzed by UHRMS on a Dionex UltiMate 3000RS (Thermo Scientific, Darmstadt, Germany) system with a charged aerosol detector interfaced with a high-resolution quadrupole time-of-flight mass spectrometer (HR/Q-TOF/MS, Impact II, Bruker Daltonik GmbH, Bremen, Germany). The Herbmix metabolome was chromatographically separated on an Acquity UPLC BEH C18 column (100 × 2.1 mm, 1.7 μm, Waters, Manchester, UK) maintained at 50°C. The mobile phase consisted of: A (0.1% formic acid in Milli-Q water, v/v) and B (0.1% formic acid in acetonitrile, v/v) at a flow rate of 0.4 mL/min. The gradient elution was: 7% B from 0 to 0.5 min with a short 0.3 min calibration segment, and the concentration of B was then increased to 70% from 0.5 to 17 min. The column was eluted with this concentration of solvent B for 0.5 min and was then re-equilibrated for 0.2 min at a flow rate of 0.5 mL/min at 50°C. The samples were kept at 15°C in the autosampler. The injection volume was 5.0 μL. The mass spectrometer was operated in the positive atmospheric-pressure chemical ionization (APCI) mode after confirmation of low sensitivity and poor resolution in the negative mode. An APCI tuning mix (Agilent, Santa Clara, CA, USA) (Pos) in quadratic mode and locked mass was used for calibration with the following parameters: capillary voltage was set at 2.8 kV, nebulizer 0.7 bar, dry gas 6.0 L/min and dry temperature 200°C. The mass scan range was set at 50-1870 m/z. MS/MS spectra were acquired in a data-dependent manner, whereby ions (maximum 2) from each scan were subjected to collisioninduced fragmentation if their absolute intensity exceeded 1800 counts. The variable collision energy ranged from 15 to 35 eV depending on the ion's m/z. Internal calibration used an APCI-TOF (pos) tuning mix introduced to the ion source via a 20-μL loop at the beginning/end of each analysis using a six-port valve. Data were collected and processed by DataAnalysis 4.3 (Bruker Daltonik GmbH, Bremen, Germany). Stock solutions of quercetrin (quercetin 3-rhamnoside), rosmarinic acid and cryptotanshinone were prepared in MeOH at concentrations of 4.2, 5.6 and 4.1 mg/mL, respectively, and kept frozen until used. Calibration curves for these three compounds were constructed based on six concentration points (from 500 ng/mL to 6 μg/mL). The concentrations of the phenolic derivatives in the Herbmix sample were calculated as equivalents of quercetrin (quercetin 3-rhamnoside) or rosmarinic acid, and cryptotanshinone was used to calculate the amount of diterpenoids identified in the extract. All analyses were performed in triplicate.

Parasitological techniques
Fecal samples were collected on D20, D28, D35, D42, D49, D56, D62 and D70 post-infection and stored at 5°C in a cooling box until laboratory examination. A modified McMaster technique [21] with a sensitivity of 50 EPG of faeces was used for the detection of strongylid eggs. All animals were humanely killed on D70 and postmortem helminthological dissections were performed. The abomasum was removed and opened, and the contents were emptied into a bucket. The abomasal mucosa was washed gently with water, washing the parasites into the bucket. The contents of the bucket were adjusted to two liters and thoroughly mixed. Two aliquots of 100 mL were then taken, and the numbers of H. contortus in each aliquot were counted.

Hematological parameters
Samples of blood were collected from the jugular vein of each animal on D7, D35, D49 and D70 using a 21-gauge needle and syringe and were placed into microtubes containing 1.6 mg/mL EDTA-K3 (Sarstedt AG & Co, Nümbrecht, Germany). Hematological parameters (red blood cells, hemoglobin, hematocrit, total leukocytes, lymphocytes, monocytes, eosinophils and basophils) were determined immediately by an automated hematological analyzer (Abbott CELL-DYN 3700, Global Medical Instrumentation, Inc., Ramsey, USA). Blood samples for sera were collected into 10-mL serum-separator tubes (Sarstedt AG & Co, Nümbrecht, Germany) and centrifuged at 1200 g for 10 min at room temperature. The sera were stored at − 80°C until analysis.

Inflammatory response
The concentrations of serum amyloid A (SAA) and serum calprotectin were determined using commercial sheep ELISA kits (MyBioSource Ltd., San Diego, USA).
Undiluted serum samples were analyzed following the manufacturer's instructions. The SAA and calprotectin concentrations had coefficients of variation < 10 and < 15% (intra-and interassay), respectively. The sensitivities of the Elisa kits were 0.1 ng/mL for calprotectin and 0.1 μg/mL for SAA. The detection range of the SAA kit was 3.12-100 μg/mL. All samples were analyzed in duplicate. The optical density of the samples was determined at 450 nm using an Apollo

Antioxidant status
The total antioxidant capacity (TAC) of the serum was measured by an assay for ferric reducing antioxidant power described by Benzie and Strain [22]. A ferrous sulfate solution was used to create a standard curve, and the results were expressed in mmol Fe 2+ formed per liter of sample. The activity of glutathione peroxidase (GPx) in the blood was assessed spectrophotometrically as described by Paglia and Valentine [23] using a Ransel kit (Randox Laboratories, Ltd., London, UK). The enzymatic activity was evaluated at 37°C at a wavelength of 340 nm, and the results are expressed in units per mL of blood. The extent of lipid peroxidation indicated by malondialdehyde (MDA) level using 1, 1, 3, 3-teramethoxypropane (Sigma-Aldrich) as an MDA precursor in the calibration curve was determined following the method of Jo and Ahn [24]. Serum total thiol or sulfhydryl (SH) concentration was determined spectrophotometrically using Ellman's method, based on the reaction of 5,5′-dithio-bis (2-nitrobenzoic acid) with protein thiol groups measured at 412 nm [25]. The concentration of SH groups was calculated using reduced glutathione as the standard, and the results are expressed in mmol/L.

Mineral status
The mineral content in the diet substrates and the serum concentrations of zinc, iron and copper in the lambs were determined by flame atomic absorption spectrometry in an air-acetylene flame, with deuterium background correction [26], using an AA-7000 atomic absorption spectrophotometer (Shimadzu Co., Kyoto, Japan). Certified lyophilized human plasma, ClinCheck Control (Recipe, Munich, Germany), was used to determine the precision of the analysis.

Calculations and statistical analysis
Calprotectin, SAA and IgA concentrations were calculated by a four-parameter logistic curve fit (GraphPad Prism, GraphPad Software, Inc., San Diego, USA). Analyses of variance (ANOVAs) (GraphPad Prism, Graph-Pad Software, Inc., San Diego, USA) were used for analyzing initial body weights (BWs), live-weight gains (LWGs), hematological parameters, inflammatory response, antioxidant status and mineral status as repeated-measures mixed models representing the four animal groups (Control, Herbmix, Zn and Herbmix +Zn) and sampling days. Effects included in the model were treatment, time and the interaction between treatment and time. Student's t-tests were applied to assess the differences between mean egg outputs (EPGs) on different sampling days and worm counts at dissection (Fig. 2, Fig. 3). Results were considered statistically significant at P < 0.05.

Bioactive compounds
Quantitative analyses of the bioactive compounds in Herbmix (  Fig. 1 represent the main compounds as numbered in Table 2.

Parasitological status
All lambs had similar BWs (Table 3). Time (P < 0.001) affected BW and the mean cumulative LWG. The patterns of shedding of eggs in C, Hmix, Zn and Hmix+Zn are shown in Fig. 2. Data from D49 were used to determine the reduction in egg output in Hmix, Zn and Hmix+Zn relative to C. The mean fecal egg count for all groups increased until D49, with no significant differences between groups (P > 0.05), but the egg counts in the lambs treated with Herbmix, zinc and a combination of both decreased after D49. Zn and Hmix+Zn EPGs were significantly lower by D56 (P < 0.05 and P < 0.01, respectively). EPGs on D70 were significantly lower in all treated groups compared to C (P < 0.01). The results of the necropsy on D70 are shown in Fig. 3. Mean worm counts 70 d post-infection were significantly lower in Hmix, Zn and Hmix+Zn than in C (P < 0.05 and P < 0.01).

Hematological parameters
The complete red blood cell (RBC) hemograms of each infected animal identified clinical signs of haemonchosis such as anemia from D35 (Table 4). RBC count, hemoglobin (HGB) level and hematocrit (HCT) were influenced by treatment (P < 0.001) and time (P < 0.001). The treatment and time affected white blood cell (WBC) levels (P < 0.05), but differential counts were not affected (P > 0.05).

Inflammatory response
Mean SAA concentration ranged from 13.7 to 23.7 μg/ mL ( Table 5). Effect of treatment and the interaction of treatment and time were observed among the groups and sampling days (P < 0.05). Mean serum calprotectin concentration ranged from 41.9 to 50.0 ng/mL. Calprotectin concentrations were not influenced by treatment, time and treatment × time (P > 0.05). Serum IgA levels were influenced by treatment (P < 0.01).

Mineral status
The serum concentrations of zinc (P < 0.001), iron (P < 0.01) and copper (P < 0.001) were influenced by time (Table 7). An interaction of treatment × time was identified for the zinc concentration (P < 0.001).

Parasitological status
The effect of Herbmix on BW and LWG in the infected lambs previously observed by Váradyová et al. [42] was not confirmed in the present experiment. Many studies have confirmed the negative impact of gastrointestinal nematode infections on sheep performance. The results of a meta-analysis indicated that weight gain in animals infected with H. contortus was 77% of the gain in parasite-free animals [43]. Most of the trials in the metaanalysis reported a negative effect of parasitism on production, but the effect was significant in only 58.3% of the trials. EPGs in the treated groups in our study decreased significantly only toward the end of the experiment, after D56. Obtaining significant LWGs in the treated groups in last 14 days of experiment was thus not surprising. Egg output began to decrease in all three treated groups after D49, with reductions > 70% between D49 and D70. Two conclusions can be drawn. Firstly, no treatment effect has been documented, because egg output did not change until D49. This is in contrast with   previous results obtained by Váradyová et al. [42] where egg output decreased by D35. However, Herbmix they used contained more medicinal herbs including also Artemisia absinthium and the anthelmintic activity of Artemisia species against the ovine nematodes has well been documented [44][45][46]. Secondly, animals increased their resistance to the worms and expelled adult parasites during the course of infection, supported by the significant reductions of parasites in the treated groups at necropsy. Significant effect of treatment on serum IgA levels pointed out on second conclusion that local antibody response may play important role to immunity of lambs to H. contortus infection.

Hematological parameters
The experimental infection of the four groups of lambs with H. contortus caused clinical signs of haemonchosis such as anemia as was described by Bordoloi et al. [47]. RBC and HGB were lower for all four infected groups than the normal ranges of 9.00-15.0 T/L and 90.0- 150.0 g/L, respectively [48]. HGB was higher in Hmix and Zn than C. Increased levels of HGB in goats fed a tannin-rich plant mixture containing condensed tannins (1.96%) were described by Jan et al. [49].

Inflammatory response
The concentrations of SAA, serum calprotectin and IgA were evaluated as markers of inflammation to monitor H. contortus infection. SAA is a non-specific inflammatory protein indicating inflammatory disease, injury or infection but is a highly sensitive, effective marker of inflammation in ruminants [50][51][52]. The effects of gastrointestinal nematodes on SAA concentration have not been extensively studied, but Ulutaş et al. [53] reported an increase in SAA concentrations with mixed gastrointestinal infections of nematodes and liver trematodes. The concentration of SAA in our study was influenced, however no effects were observed in our previous study of lambs infected with H. contortus [42]. The response of SAA to the experimental conditions was weak and non-uniform, so we cannot currently consider SAA a useful marker for monitoring H. contortus infections in  lambs. Calprotectin is a major cytosolic protein of leucocytes, especially neutrophils. Elevated levels of calprotectin are evident in infectious and inflammatory diseases and are often used as a marker of gastrointestinal inflammation [54]. The response of serum calprotectin during H. contortus infection and the experimental treatments in our present study was inconsistent with study of Váradyová et al. [42]. Serum IgA in sheep is predominantly derived from the intestine and closely associated with intestinal mucosal immune responses [14]. Cardia et al. [55] reported higher serum IgA levels against L3 in lambs infected with Trichostrongylus colubriformis. Increased levels of IgA in naturally infected sheep have been positively associated with resistance to Teladorsagia circumcincta by suppressing parasite growth; development and fecundity, mediated by IgA activity against L4 larvae [56,57]. Serum IgA levels in our study were influenced by treatment, however the immune response against H. contortus was very similar in all experimental groups.

Antioxidant status
Several studies have reported that medicinal plants have a wide range of antioxidant capacities and that phenolic compounds are a major contributor to the antioxidant activity of these plants, making them promising sources of natural antioxidants [58]. Herbmix containing predominantly flavonoids (54%) exhibited antioxidant potential in vivo [7] by reducing the MDA level, indicating a decrease in lipid peroxidation in the serum, and tending to increase GPx activity in the blood of infected lambs. Zinc can exert its antioxidant action by several possible mechanisms, e.g. the protection of protein SH groups from oxidation. Zinc is also involved in the synthesis of molecules rich in SH groups, such as reduced glutathione and metallothionein, which play an antioxidative role [59]. The levels of SH groups on D49 and D70 indicated that zinc supplementation improved the protection of various thiols in the infected lambs by increasing serum SH levels. The activity of blood GPx was higher, and lipid peroxidation in the serum was lower, at the end of the experiment in the groups treated with zinc than in the untreated group. Pivoto et al. [60] also demonstrated that zinc could help reduce the oxidative stress caused by H. contortus in lambs. Our findings indicate that treatment with organic zinc alone or in combination with herbal nutraceuticals could potentially provide natural antioxidants for minimizing oxidative stress in nematode infected lambs.

Mineral status
Experimental infection with H. contortus can decrease mineral concentrations in the liver or serum of lambs [42,61]. Concentration of serum zinc of lambs in our treatments was not influenced. The efficiency of immunological responses against gastrointestinal nematodes depends on the zinc intake of the host, supporting the important role of zinc in the maintenance of the gut epithelial barrier and intestinal immunity [62]. Our unsupplemented control lambs had marginal serum levels of iron, associated with their low blood HGB levels, but supplementation with zinc or Herbmix maintained higher serum zinc and iron levels, providing better conditions for the lambs to cope with this parasitic disease and reduce parasitic burdens.

Conclusion
This study did not fully confirm a direct anthelmintic impact on the viability of nematodes, but the treatments with herbal nutraceuticals, zinc and both diet supplements together likely indirectly contributed to an increase in the resistance of lambs to nematode infections. The bioactive compounds identified in Herbmix have anti-inflammatory and antioxidant biological activities with beneficial health-promoting effects. The Herbmix, organic zinc and combination of Herbmix with organic zinc contributed to reduction of nematode parasitic infection in experimental animals. The use of herbs with bioactive compounds together with some essential trace elements as alternatives to conventional anthelmintics can be useful for economical, effective and sustainable animal production.