Immunomodulatory effects of Echinacea and Pelargonium on the innate and adoptive immunity in calves

ABSTRACT Immunomodulatory effects of Echinacea purpurae and Pelargonium sidoides in calves were investigated. Sixty 25-day-old nonvaccinated calves living in integrated farm unit were randomly selected and were divided into 5 treatment groups consisting of 12 calves each; 4 groups were receiving different amounts and/or times of Echinacea and Pelargonium and the control group received placebo. Blood levels of γ-interferon, cytokine gene expression, lactoferrin and total IgG were analysed on days 0, 9, and 30. When levels for total IgG, γ-interferon, mRNAs for the γ-interferon, IL-1-β, IL-4 and the tumour necrosis factor-α genes were compared from days 0 to 9 post-treatment, significant differences were found between treated and non-treated calves using various amounts of Echinacea and Pelargonium; a doses of 250 mg Echinacea and 62.5 mg Pelargonium for 5 days seems to be ideal. Echinacea purpurae and Pelargonium sidoides are able to modulate immune functions in calves.


Introduction
Calf health and its management are important for the economy and productivity of cattle operations. Calves are exposed to many stress factors such as transport, inadequate feeding, constant exchange of animals, overcrowding, improper climate and poor hygienic conditions. These stress factors cause a decrease in natural immunity which in turn allows access of pathogens mainly through mucosal sites. Since the calf's immune system is not well established in the first weeks of life, adequate colostral supply and good husbandry are critical to avoid intestinal and respiratory pathogens (Ayrle et al., 2016;Cho & Yoon, 2014;Lorenz et al., 2011). In order to improve calf health and immunity, numerous prophylactic tools such as antibiotics, vaccination, biosecurity and good husbandry are applied (Ayrle et al., 2016;Windeyer et al., 2014;Wu, He, & Kang, 2017).
Phytotherapy is an alternative health tool since it is able to modulate the innate and adaptive immunity with positive results for herd health. In addition, herbal products are advantageous to certain antibiotics since they do not promote bacterial resistance (Theisen & Muller, 2012). In order to reduce the antibiotic usage in farm animals, products derived from medicinal plants are used worldwide for the prevention and treatment of infectious diseases in animals and humans. However, the effects of many medicinal herbs on the immune system in animals have not been investigated in detail (Ayrle et al., 2016).
There are several reports on the effects of Echinacea, Pelargonium extracts in the immune system in animals and humans. Among the nine different Echinacea species, Echinacea pallida, Echinacea angustifolia and Echinacea purpurea have generally been used as herbal medicine (Zhai et al., 2007a). Various animal studies suggest that Echinacea may modulate innate and adaptive immune responses. It has previously been shown that E. purpurea activates murine/human macrophages by inducing the production of interferon (IFN) and interleukin (IL)-1 (Rininger, Kickner, Chigurupati, McLean, & Franck, 2000). Echinacin, derived from E. purpurea, has been reported to attach to macrophages and T cells, which protects the host from viral invasion (Goel et al., 2002;Xiong, Kadota, Tani, & Namba, 1996). In addition, a polysaccharide derived from Echinacea inhibited fungal and bacterial growth in mice and increased the level of reactive oxygen intermediates and macrophage chemotaxis as well as production of IL-10, IL-6, IL-1-β and tumour necrosis factor-α (TNF-α) (Roesler et al., 1991;Steinmuller et al., 1993). However, ethanol extracts of Echinacea decreased monocyte and macrophage responses in mice to lipopolysaccharide and endotoxin by downregulating the production of TNF-α and prostaglandin E2 (LaLone et al., 2007;Stevenson et al., 2005). In another study mice, E. angustifolia enhanced bacterial killing while E. pallida and E. purpurea did not have an effect on killing (Zhai et al., 2007b).
Cytokines play an important role in progression and prevention of infectious diseases by modulating the immune response such as induction of fever, the production of cellular and humoral factors such as antibodies, cytokines, acute phase proteins and interferons. Therefore, it is important to understand cytokine expression levels in immune cells in order to predict the innate and adaptive immune response to different pathogens after infections and vaccinations (Konnai, Usui, Ohashi, & Onuma, 2003;Pacheco et al., 2015;Prohl et al., 2015). The measurement of cytokine expression in blood cells is an important tool and real-time RT-PCR, ELISA, Northern Blotting and flowcytometry are generally used to determine the level of cytokine expression (Brodzki, Kostro, Krakowski, & Marczuk, 2015;Konnai et al., 2003;Pacheco et al., 2015;Prohl et al., 2015). Many studies to measure cytokine expression in humans using real-time RT-PCR was performed (Brendler & van Wyk, 2008;Dapas et al., 2014;Senchina et al., 2006), whereas only a few studies investigated the level of cytokine expression in cattle after administering phytotherapy (Ayrle et al., 2016;Wu, Nardone, & Lacetera, 2009).
Pelargonium sidoides and Echinacea extracts are frequently used for the treatment of certain viral and bacterial infections in humans and it has been demonstrated by many researchers that these herbs improve the innate and adaptive immunity. However, the effects of these herbs on calf health and immunity are not fully understood. Therefore, this study was designed to investigate the effects of Echinacea and Pelargonium on the cytokine gene expression, total IgG, γ-interferon and lactoferrin levels in calves.

Herbal preparation and study animals
In this study, a tablet form of the herbal medicinal product (CEVASTIM Tablet) containing the equivalent quantity of the ampoule formulation of Echinacea purpurae (500 mg) and Pelargonium sidoides (135 mg) was used orally.
The study consisted of the 25 days old, Holstein, nonvaccinated calves, living in a good husbandry conditions in an integrated farm unit located in the Thrace district of Turkey. Randomly selected calves were divided into 5 animal groups (4 treatment groups and 1 control) consisting of 12 calves in each group. Vaccination status was considered in dividing groups. For this, one control nonvaccinated, one vaccinated (½ tablet for 5 days), and three nonvaccinated groups (½ tablet for 5 days, 1 tablet for 5 days, ½ tablet for 10 days) were designed. The vaccinated and nonvaccinated groups received the treatment (CEVAS-TIM tablet) orally while control receiving placebo (Table 1).
All calves received the same feed and husbandry conditions like the other calves on the farm. The animal groups, amount of product administered, sample collection and study scheme are shown in Table 1. Blood was collected in tubes with EDTA for determination of γ-interferon by ELISA, in TEMPUS Blood RNA tubes (Applied Biosystems, Cat No. 4342792) for evaluation of cytokine gene expression and in serum tubes to obtain sera for Lactoferrin and total IgG analyses as indicated in Table 1.
Determination of γ-interferon, lactoferrin and total IgG levels by ELISA γ-interferon levels in blood were determined by using a commercial test kit (AniGen TBferon, Bionote, 3801P0001) as described by the manufacturer. Serum lactoferrin and total IgG levels were also determined using commercial test kits as described by the manufacturers (BETHYL, Bovine Lactoferrin-ELISA Quantitation Set, E10-126, and (Bovine IgG-ELISA Quantitation Kit, BETHYL, E10-118). All sera were analysed in duplicates.

RNA extraction and reverse transcription
Total RNA was extracted from blood samples by using the Tempus Spin RNA Isolation Kit as described by the manufacturer (Thermo Fisher Scientific, Cat No. 4380204). The amount of RNA in the extracted material (90 µl) was measured using a NanoDrop spectrophotometer (NanoDrop 1000c, Thermo Scientific, Waltham, MA, USA). The extracted RNA (about 300 ng) was used for cDNA synthesis.
Reverse transcription and generation of cDNA were performed with 10 µl RNA using a high-capacity Reverse Transcription Kit (ThermoFischer Scientific, Cat No: 4368813). Twenty (20) microlitre of cDNA was generated according to the manufacturer's instruction.

Data and statistical analyses
For the gene expressions study, to control suitability of selected internal control gene, PCR efficiency was calculated for cytokine genes and β-actin gene by tenfold serial dilution of cDNA. A plot was generated with the C T (y-axis) versus log cDNA dilution (x-axis) and slope was determined. PCR efficiency (E) was calculated by the equation slope = −(1/LogE). The mRNA expression was analysed triplicate for each cytokine genes and β-actin gene. Delta C T (ΔC T ) was calculated between each cytokine gene and β-actine gene and fold changes between treated and control groups was calculated using 2 −ΔΔCT formula (Schmittgen & Livak, 2008). The P-value (P < .05) were determined based on Student's t-test for checking internal control gene (β-actine) changes under the experimental conditions and between ΔC T values for control and treatment groups. Student's t-test was performed using GraphPad Prism version 7.00 (Yuan, Reed, Chen, & Stewart Jr, 2006).

Lactoferrin levels
Lactoferrin levels varied amongst experimental and control animals. The highest OD obtained after day 9 was 1590 ( Figure 2(A,B)). The increase in lactoferrin levels from days 0 to 9 in all treatment groups was not statistically significant. However, a decrease seen in animals ½ tablet administered for 5 days (ID 21-30) was statistically significant.

Total IgG levels
Total IgG levels varied amongst experimental and control animals. The highest OD obtained after day 9 was 0.900 ( Figure 3(A,B)). The difference in total IgG levels between the days 0 and 9 was statistically significant only in animals given ½ tablets for 5 days (ID 21-30; Figure 3(A,B)). There was an increase in the total IgG level in animals received ½ tablet for 5 days vaccinated (ID 1-10), but this was statistically not significant (P = .08).
β-actin and cytokine gene expression PCR efficiency of the β-actin and cytokine genes were found to be between 1.8 and 2.2 which is sufficient for comparison. The mean and 95% of confidence intervals of fold changes for all cytokine genes are given in Table 2 and Figure 4. Results of the statistical analyses of the delta C T values between reference and target (cytokine) genes for both control and treatment groups are given in Table 3. There were no significant changes in β-actin gene expression before (day 0) and after treatment (day 9) except the group of animals received ½ tablet treatment for 10 days (Table 3). The results of the animal groups in target gene expression are explained below.
Animal treatment groups for cytokine gene expression ½ tablet 5 days-vaccinated group Interferon γ, IL-1-β and IL-2, IL-4 and TNF-α gene expression were upregulated in the animals treated with ½ tablet product for 5 days and received routine vaccination Figure 1. (A,B) OD of γ-interferon levels of all groups before (day 0) and after (day 9) oral administration. Animal ID: from 1 to 10 -½ tablet for 5 days vaccinated; from 11 to 20controls; from 21 to 30 -½ tablet for 5 days; from 31 to 40 -1 tablet for 5 days; from 41 to 50 -½ tablet for 10 days. Light bars: day 0; dark bars: day 9.
(Table 2, Figure 4). The gene fold expression calculated by using the values in control (day 0) and after (day 9) treatment for interferon γ, IL-1-β, IL-2 and TNF-α were statistically significant (Table 3). Changes in control groups were not statistically significant (Table 3).
½ tablet for 5 days-nonvaccinated group Interferon γ, IL-1-β and IL-2, IL-4 and TNF-α gene expression were upregulated in the animals treated with ½ tablet product for 5 days (Table 2, Figure 4). The gene fold expression calculated by using the values in control (day 0) and after (day 9) treatment for interferon γ, IL-1-β, IL-2 and TNF-α were statistically significant (Table 3). Changes in control groups were not statistically significant (Table 3).

tablet for 5 days-nonvaccinated group
Interferon γ, IL-1-β and IL-2, IL-4 and TNF-α gene expression were upregulated in the animals treated with 1 tablet product for 5 days (Table 2, Figure 4). The gene fold expression calculated by using the values in control (day 0) and after (day 9) treatment for IL-1-β, IL-2 and TNF-α were statistically significant (Table 3). Changes in control groups were not statistically significant (Table 3).
expression calculated by using the values in control (day 0) and after (day 9) treatment for interferon γ, IL-1-β, IL-2, IL-4 and TNF-α was statistically significant (Table 3). Changes in control groups were also statistically significant (Table 3). Therefore, the upregulation in the treated group cannot be concluded as upregulation.

Summary of the comparison of all groups for all parameters analysed
Summary of the statistically significance and nonsignificance for all parameters are shown in Tables 3 and 4. The increase in γ-interferon levels on day 9 was statistically significant in groups received ½ tablet for 5 days-vaccinated, ½ tablet for 5 days and ½ tablet for 10 days ( Table 4). The difference in total IgG levels between the days 0 and 9 was statistically significant only in animals given ½ tablets for 5 days (Table 4). However, lactoferrin levels were decreased in all groups (Table 4).
For the cytokine gene expression; in ½ tablet 5 days-vaccinated group, the gene fold expression for interferon γ, IL-1-β, IL-2 and TNF-α was statistically significant (Table  3). In ½ tablet for 5 days-nonvaccinated group, the gene fold expression for interferon γ, IL-1-β, IL-2 and TNF-α was statistically significant (Table 3). In 1 tablet for 5 days-nonvaccinated group, the gene fold expression for IL-1-β, IL-2 and TNF-α was statistically significant (Table 3). In ½ tablet for 10 days-nonvaccinated group, the gene fold expression for interferon γ, IL-1-β, IL-2, IL-4 and TNF-α was statistically significant in both study group and control group (Table 3). Therefore, the upregulation in this group cannot be calculated and therefore cannot be considered to be as upregulation.

Discussion
Respiratory and mucosal pathogens have a significant impact on the cattle health (Cho & Yoon, 2014;Pfützner & Ozsvari, 2017;Windeyer et al., 2014). Neonatal calf diarrhoea and respiratory infections are found to be the most frequent cause of calf losses with a mortality up to 55% in many countries (Ayrle et al., 2016Bähler et al., 2012Cho & Yoon, Table 3. Summary of the results from the statistical analyses of the delta C T values between reference and cytokine genes for both control and treatment groups. NS: statistically not significant; S: statistically significant (P < .05).  2014; Lorenz et al., 2011;;Meganck, Hoflack, & Opsomer, 2014;Windeyer et al., 2014). Vaccines, antibiotics, biosecurity and good husbandry are generally applied to combat those infections in calves. In addition to these, alternative approaches like herbal products are needed to promote calf immune system to combat pathogens in calves. In contrast to human and experimental animals, there are not many studies related to use of phytotherapy to investigate immunomodulatory effect in farm animals (Basmacioğlu Malayoğlu et al., 2010;Ayrle et al., 2016;Baruh & Kacabağlı, 2017). In the absence of comprehensive information on the effect of Echinacea and Pelargonium in cattle immunity, this study was performed to investigate potential efficacy of Echinacea and Pelargonium on the cytokine expression, total IgG, γ-interferon and lactoferrin levels in calves. Up to date, the effect of Echinacea, Ginseng and Tinospora on the immune modulation of cattle to pathogens have been investigated (Hu, Concha, Lin, & Persson Waller, 2003;Mukherjee, De, & Ram, 2010;Wu et al., 2009). In one study, extract of Echinacea angustifolia increased the PBMNC count and interferon production in cattle (Wu et al., 2009). In another study, using the Ginseng extract in cattle with mastitis has formed an adjuvant effect when combined with ovalbumin and Staphylococcus aureus bacterin by increasing lymphocyte count and the antibody response (Hu et al., 2003). In the other study, in cattle with mastitis, the extract of Tinospora cordifolia has increased the somatic cell count, phagocytic and lysosomal enzyme activity and IL-8 level, and caused reduction of the total bacterial count in milk (Mukherjee et al., 2010). Results of these studies indicated that medicinal herbs have potential to modulate immune system in cattle.
In the present study, using the Echinacea and Pelargonium extract in calves have increased total IgG level, γ-interferon level and cytokine gene expression. Echinacea also caused an increase in immunological parameters in dogs and sheep. In dogs which received Echinacea formulation, PCV, Hb, RBC count, WBC count, Lym, Nut, the per cent of phagocytosis and IgM level were significantly increased (P < .05) (Torkan, Khamesipour, & Katsande, 2015). In sheep, Salmonella enteritidis (SE) bacterin and dried roots of Echinacea purpurea (EP) were used together and enhancement in antibody production was reported (Barbour et al., 2015).
Species of an herb, the amount of the product, duration of administration and the method analysed will effect the immunomodulation of the medicinal herbs in a given animal (Ayrle et al., 2016). In the present study, Echinacea and Pelargonium were used since the results of previous studies have shown that these herbs modulate the immune system (Ayrle et al., 2016;Barrett, 2003;Brendler & van Wyk, 2008;Kayser, Kolodziej, & Kiderlen, 2001). The amount and duration of these herbs were tested in this study by using a different amount of CEVASTIM in different durations. The method, real-time RT-PCR to estimate cytokine expression levels used in this study was also used by many investigators (Dapas et al., 2014;Konnai et al., 2003;Puech, Dedieu, Chantal, & Rodrigues, 2015). Results of previous studies have indicated that cytokine gene expression usually increases after 72-96 hours of immunization (Dar, Hajam, Suryanarayana, Kishore, & Kondabattula, 2015). Therefore, in the present study, blood was taken to measure cytokine gene expression level 96 hours after stopping oral administration of CEVASTIM.
In contrast to calves, immunomodulatory effect of Echinacea and Pelargonium has been widely investigated in human and experimental animals (Agbabiaka et al., 2008;Moyo & Van Staden, 2014;Uluısık & Keskin, 2012;Wagner et al., 2015). It has been demonstrated that extract of both plants especially Pelargonium extract (EPs 7630) have anti-infective properties (Brendler & van Wyk, 2008;Conrad & Frank, 2008;Timmer et al., 2013). In a study performed by Hall and others (2007), Echinacea purpurea reduced the duration of upper respiratory infections in human. In influenza vaccinated human, four Echinacea species (E. angustifolia, E. purpurea, E. simulata, E. tennesseensis) had no effect on IFN-γ production, diminished the IL-2 production but augmented the IL-10 production. However, Echinacea pallida suppressed expression of all cytokines (Senchina et al., 2006). In the present study, in treatment groups of animals, Echinacea and Pelargonium combination had no effect on IL-4 gene upregulation but upregulated the other targeted cytokine genes except the group which received ½ tablet fro 10 days. In one study, Echinacea was found to have anti-inflammatory properties since reduced severity of symptoms have been seen in vivo during the treatment of upper respiratory tract infections with Echinacea (Fast, Balles, Scholten, Mulder, & Rana, 2015). However, TNF-α production was decreased. In the present study, the TNF-α gene expression was upregulated in 3 treated groups (1/2 tablet for 5 days vaccinated, ½ tablet for 5 days and 1 tablet for 5 days) when compared to controls. In another study in human, a syrup containing Echinacoside and polysaccharide from Echinacea was administered to 10 people once a day for 1 month. Results showed that IL-2 and IL-8 were upregulated while the mRNA levels of the pro-inflammatory cytokines TNF-α and IL6 downregulated. This regulation was correlated with the cytokine levels in plasma (Dapas et al., 2014). In the present study, similar results were obtained that IL-2 expression was upregulated except in a group of animals received ½ tablet products for 10 days. In one study, the efficacy of 5 days of Pelargonium sidoides therapy for preventing asthma attack during upper respiratory tract viral infections was investigated. Sixty-one asthmatic children with upper respiratory tract viral infection were enrolled in the study which indicated the children administered Pelargonium sidoides, had less frequency of asthma attack (Tahan and Yaman, 2013). Similarly, in the present study, the 5 days regime had a better effect on targeted cytokine gene expression in calves except IL-4. In another study in human, EPs® 7630 (100 μg/ml) interfered with replication of seasonal influenza A virus strains (H1N1, H3N2), human coronavirus, respiratory syncytial virus, parainfluenza virus, and coxsackie virus but did not have any effect on the replication of highly pathogenic avian influenza A virus (H5N1), rhinovirus or adenovirus (Michaelis, Doerr, & Cinatl, 2011).
The EPs-7630 experiments with regard to cytokine expression gene (iNOS, IFN-α, IFNγ, TNF-α, IL-1, IL-10, IL12, IL-18) have shown the immunomodulatory effect of Pelargonium in response to infected macrophages (Kolodziej & Kiderlen, 2007). Similar results were obtained in experimental animals considering the anti-infective and immunomodulatory effect of Echinacea and Pelargonium. In one study, mice were administered once a day (for 7 days) with Echinacea extracts and immunized with sheep red blood cells (sRBCs). Antibody response to sRBC was significantly increased by three Echinacea species. They caused a significant increase in interferon-α production, but inhibited the release of TNF-γ and IL-1-β. The inhibition of TNF-α was also seen in this study except in one study group. E. angustifolia and E. pallida administered mice demonstrated significantly higher production of IL-4 and IL-10. These results indicated that Echinacea modulates both innate and adaptive immune responses (Zhai et al., 2007a). In a murine model, E. purpurea activated macrophages to stimulate IFN-γ production (Mishima et al., 2004).
In a study performed in rats by using Echinacea and Ginseng, an increase in expression of IL-10 and TNF-α genes were found (Uluısık & Keskin, 2012). In mice, IMMUNAL was found to be effective in enhancing antibody production to sRBCs but no such effect was seen when ECHINACEA FORTE drops used (Bałan, Sokolnicka, Skopińska-Różewska, & Skopiński, 2016). In one study, all the Pelargonium extracts (EC(50), gallic acid (EC(50) and its methyl ester (EC(50) significantly decreased the intracellular survival of L. donovani amastigotes within murine macrophages by triggering macrophage activation (Kayser et al., 2001). As a murine model of tuberculosis, Pelargonium sidoides stimulated macrophage killing, but not uptake of M. tuberculosis and M. fortuitum equally well (Kim, Griffiths, & Taylor, 2009).
Animal experiments have shown that a combination of Echinacea extracts demonstrated greater effects than extracts of a single plant (Bodinet et al., 2002). Therefore, two plants, Echinacea and Palergonium were used in this study. However, using a mixture of plants makes it difficult to distinguish the biological significance of each plant or identify redundancy in effects (Bodinet et al., 2002). Results of this study, combination of both plants were tested, indicated that this combination resulted in increase in γ-interferon levels in three groups of treated animals, and total IgG level in a group of treated animals which received CEVASTIM ½ tablet for 5 days as well as upregulation of targeted cytokine gene expressions except IL-4.
Interesting part of this study was that ½ tablet of the extract used for 10 days induced upregulation of cytokine gene expression but upregulation was also seen in the controls and therefore cannot be concluded as positively correlated with fold changes. This might be associated with several factors such as duration and dose of the extract used. This effect is most likely associated with duration since an increase was observed in all targeted cytokine gene expression, total IgG and γ-interferon levels when CEVASTIM used for ½ tablet for 5 days. All of these effects were not seen in other treated animal groups while some groups were partly responded.

Conclusion
Using ½ tablet CEVASTIM in calves for 5 days has a potential to induce natural immune response as a result to modulate innate and adoptive immunity by increasing IgG and γinterferon levels and upregulation of important cytokine (γ-interferon, IL-1-β, Il-2 and TNF-α) gene expression in calves. Summary of the statistically significant increase in ELISA and real-time PCR gene expression results are illustrated in Figure 5.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This study was supported by CEVA Animal Health and the University of Istanbul, Veterinary Faculty Istanbul.