Amelioration of Ochratoxin A-induced immunotoxic effects by silymarin and vitamin E in White Leghorn cockerels

Abstract

Silymarin (SL) is the bioactive extract of the plant Silybum marianum and Vitamin E (VE) is an important anti-oxidant. The present study was designed to evaluate potential ameliorative effects of SL and VE against Ochratoxin A (OTA)-induced immunotoxic effects in White Leghorn cockerels. One day-old birds were divided into 12 groups (20 birds/group) and fed basal diets amended with OTA (1.0 or 2.0 mg/kg) alone or in combination with SL (10 g/kg) and/or VE (200 mg/kg) for 42 days. Immunological in situ responses, including antibody formation against sheep red blood cells (7 and 14 days after both primary and booster injections), lymphoproliferative responses to avian tuberculin (30 days of age), and mononuclear phagocytic system function (i.e. by clearance of injected carbon particles) assay (42 days of age), were assessed. Results suggested that silymarin and Vitamin E alone or in combination ameliorated the immunotoxic effects induced by 1.0 mg OTA/kg but could not significantly impact on the effect from ingestion of 2.0 mg OTA/kg. The results of the present study suggested that both SL and VE possess an ability to ameliorate OTA-induced immunotoxicity in chicks. However, it remains to be determined whether/what SL:OTA or VE:OTA ratios are required to assure such mitigation of OTA-induced immunotoxicities.

Keywords::

• Ochratoxin A
• immunology
• silymarin
• Vitamin E
• White Leghorn

Introduction

Ochratoxins are fungal metabolites that are produced by different species of Aspergillus and Penicillium (Cieglar et al., 1972; Hesseltine et al., 1972; Khan et al., 2010; Hassan et al., 2012). Among the different forms of ocharatoxins, Ochratoxin A (OTA) is the most toxic (Sholesberg et al., 1997). OTA has teratogenic, mutagenic, and immunotoxic effects (O’Brien and Dietrich, 2005). It causes severe immunosuppression in situ by reducing the number of cells in lymphoid organs, inducing a reduction in antibody responses, causing a decrease in activity of natural killer (NK) cells, and affecting a reduction in the bacteriolytic potential of macrophages (Stoev et al., 2000, 2002; Alvarez-Erviti et al., 2005; Politis et al., 2005).

Silymarin (SL) is a bioactive extract of polyphenolic flavonoids of the milk thistle plant (Silybum marianum). This plant has been reported in different regions of Pakistan, including the Margalla Hills of Islamabad (Shinwari et al., 2007). SL has been reported to stimulate body immune status (Wilasrusmee et al., 2002a). It inhibited the OTA induced tumor necrosis factor (TNF)-α release from isolated Kupffer cells and the perfused liver of rats (Al-Anati et al., 2009). It also has been to ameliorate the toxic effects of aflatoxin B₁ (AFB₁) in broiler chicks (Tedesco et al., 2004). Vitamin E, an important anti-oxidant, possesses the ability to increase cell-mediated response, number of antibody producing cells, and antibody titers in chickens, mice, and buffalo calves (Meydani, 1995; Hossain et al., 1998; Qureshi et al., 2010; Ajakaiye et al., 2011). Vitamin E and silymarin, when given together, provided immunoprotective and immunostimulatory effects in male Wistar rats (Horvath et al., 2001).

Ingestion of OTA-contaminated feeds has been known to induce immunotoxic effects in chicken and other animals (Huff et al., 1974; Singh et al., 1990; Muller et al., 1995; O’Brien and Dietrich, 2005). In the literature, no reports have yet described the potential amelioration of OTA-induced immune-toxicity by providing dietary supplements to these hosts, a method that could be much more effective and practical to farmers/ranchers than methods to detoxify the contaminated feed stocks themselves. Thus, the present study was designed to first induce ochratoxicosis in White Leghorn cockerels and then to evaluate the amelioration of OTA-induced immunotoxic effects by dietary supplementation with silymarin and Vitamin E, two common and relatively inexpensive products readily available to the public.

Materials and methods

Production of OTA

OTA was produced from Aspergillus ochraceus (CECT 2948) following the method of Trenk et al. (1971), with slight modifications. The Aspergillus was originally obtained from the Culture Collection Centre (University De Valencia, Valencia, Spain) and maintained at the Department of Pathology in the University of Agriculture (Faisalabad, Pakistan). Briefly, in each of 10 1-L Erlenmeyer flasks, 80 g of wheat grain was soaked in 80 ml distilled water for 2 h and then autoclaved. An inoculum prepared from a 2-week-old A. ochraceus slant culture was mixed with the autoclaved grain, and the flask then incubated in the dark at 28°C for 14 days; each flask was shaken twice daily to break mycelial growth. After 14 days, the flasks were autoclaved and OTA was extracted from the fermented wheat using acetonitrile:water (60:40, v/v). The extract obtained was then passed through an OTA immunoaffinity column (Vicam, Watertown, MA) and OTA was eluted from the immunoaffinity column using ethanol. OTA was quantified using an LC20AT Prominance HPLC system (Shimadzu, Kyoto, Japan) equipped with an RF-10AXL fluorescence detector (Shimadzu), following the method described by Bayman et al. (2002), and the OTA concentration in the fermented wheat calculated.

Experimental design

A corn soy-based feed (21% protein, 2900 Kcal/kg metabolizable energy) free from toxin binders and coccidiostat was used as basal feed. The AFB₁ and OTA concentrations in the basal feed were verified to each be < 1 ng/g. Experimental feeds were then prepared by spiking the basal feed with fermented/OTA contaminated wheat grains, SL (Panjin Tianyuan Pharmaceutical Co., Gaosheng Town, China), and/or VE (Roche, Nutley, NJ).

A total of 240 1-day-old White Leghorn (WL) cockerels were procured from a local commercial hatchery and verified to be free from Salmonella and Mycoplasma infection. All birds were kept in battery cages in the Department of Pathology. The birds were then placed on basal feed for 2 days. Thereafter, the birds were sub-divided into 12 groups (20 birds/group) and offered diets containing different combinations of OTA (1 or 2 mg/kg), SL (10 g/kg), and/or VE (200 mg/kg) (Table 1) up until they reached 42 days-of-age. All of the animal experiments were conducted according to the rules and regulations of the Animal Ethics Committee at the University of Agriculture (Faisalabad).

Table 1.  Layout of the experiment.

Groups	No. of birds	Treatment in feed
Control	20	Nil
OTA1	20	OTA (1 mg/kg)
OTA2	20	OTA (2 mg/kg)
Silymarin (SL)	20	Silymarin (10 g/kg)
Vitamin E (VE)	20	Vitamin E (200 mg/kg)
OTA1+SL	20	OTA (1 mg/kg) + Silymarin (10 g/kg)
OTA2+SL	20	OTA (2 mg/kg) + Silymarin (10 g/kg)
OTA1+VE	20	OTA (1 mg/kg) + Vitamin E (200 mg/kg)
OTA2+VE	20	OTA (2 mg/kg) + Vitamin E (200 mg/kg)
VE+SL	20	Vitamin E (200 mg/kg) + Silymarin (10 g/kg)
OTA1+SL+VE	20	OTA (1 mg/kg) + Silymarin (10 g/kg) + Vitamin E (200 mg/kg)
OTA2+SL+VE	20	OTA (2 mg/Kg) + Silymarin (10 g/kg) + Vitamin E (200 mg/kg)

OTA1, group receiving 1 mg/kg OTA in feed; OTA2, group receiving 2 mg/kg OTA in feed; SL, Silymarin; VE, Vitamin E.

Parameters studied

Antibody responses to sheep red blood cells

For this study, a sub-set of 10 birds/group was utilized and the method described by Delhanty and Solomon (1996) was followed. Briefly, sheep red blood cells (SRBC; obtained from sheep maintained by the Department of Microbiology at the University of Agriculture (Faisalabad, Pakistan)) were washed with normal saline solution and a 3% suspension was prepared. One milliliter of this suspension was then injected intravenously as a primary dose to five birds in each group (at 13 days-of-age). Blood was collected 7 and 14 days after the primary dose. A booster dose was given 14 days after the primary injection and blood was collected again 7 and 14 days later. In all cases, serum was separated, heat-inactivated (56°C, 30 min), and titrated for total and mercaptoethanol (ME)-resistant (IgG) anti-SRBC antibody titers. ME-sensitive (IgM) antibodies titers were obtained by subtracting the level of ME-resistant antibodies from that of the total antibodies measured. All data were expressed in terms of (log₂).

Lymphoproliferative response to avian tuberculin

For this study, a sub-set of five birds/group was utilized. This test was performed on the birds at 30 days-of-age following the method of Akhter et al. (2008). Birds were randomly selected from each group and 0.2 ml tuberculin (Veterinary Research Institute, Lahore, Pakistan) was injected into the inter-digital space between the 3^rd and 4^th digit of the right foot. Phosphate-buffered saline (pH 7.4, 0.2 ml) was injected between the 3^rd and 4^th digit of the left foot as a control. The thickness of the intra-digital space was then measured at 0, 24, 48, and 72 h post-injection using a constant tension micrometer (Global Sources, Shanghai, China) to evaluate the cell-mediated immune response. The thickness response was calculated and expressed (mm) as: (right foot skin thickness − left foot skin thickness) at each timepoint.

Mononuclear phagocytic system function assay (carbon clearance assay)

For this study, a sub-set of five birds/group was utilized. The phagocytic potential of chick circulatory macrophages was assayed according to a modified method of Sarker et al. (2000). Black India ink (Pelikan 4001, Pelikan, Sharjah, UAE) was centrifuged (3000 x g, 30 min) and the supernatant collected. This material was then intravenously injected into the right wing vein (at rate of 1 ml/kg body weight) in these birds of 42 days of age randomly selected from each group. Blood samples of 200 µl were then collected from each bird before (0 min) and 3 and 15 min after injection of the ink. Blood was immediately shifted into tubes containing 4 ml of 1% sodium citrate solution. After centrifugating the samples at 50 x g for 4 min, the relative amount of carbon particles in the supernatant was estimated by measures at 640 nm in a U-2001 spectro-photometer (Hitachi, Tokyo, Japan). The absorbance from the ‘0 min’ sample was considered as the ‘zero’ value for each respective sample. Phagocytic index in the clearance assay was calculated by the formula K = (log_n OD₁ − log_n OD₂)/(T₂ − T₁), wherein OD₁ and OD₂ are optical densities at times T₁ and T₂, respectively (Juvekar et al., 2009). Thus, for the 3 min sample, T₁ = 0 and T₂ = 3; for the 15 min sample, T₁ = 0 and T₂ = 15. Any decrease in phagocytic index was deemed to be indicative of a reduction in in situ phagocytic function of resident macrophages (and vice-versa).

Statistical analysis

Using a statistical software package (MSTATC, East Lansing, MI), data obtained for all immunologic parameters were subjected to a one-way analysis of variance (ANOVA) test. For each parameter, the means of the different groups were compared using a Duncan’s multiple-range test. Values were considered significantly different at p ≤ 0.05.

Results

Antibody titers against sheep red blood cells (SRBC)

Antibody titers against SRBC in chicks fed different levels of OTA—with or without silymarin (SL) and Vitamin E (VE)—are presented in Table 2. At Day 7 post-primary SRBC injection, total antibody titers (total Ig) in birds in the SL, VE, SL+VE, OTA1+SL, and OTA1+SL+VE groups were not significantly different from control values. Titers in birds in groups OTA1, OTA1+VE, OTA2, OTA2+VE, OTA2+SL, and OTA2+SL+VE were significantly lower than control. IgG titers in all groups except for OTA2+VE, OTA2+SL, and OTA2+ VE+SL showed no significant differences from the controls.

Table 2.  Antibody titers (log2) against SRBC at different days.

Groups	Time after primary SRBC injection				Time after booster SRBC injection
	7 days		14 days		7 days		14 days
	Total Ig	Ig G	Total Ig	Ig G	Total Ig	Ig G	Total Ig	Ig G
Control	2.99 ± 0.21^a	2.51 ± 0.23^a	2.70 ± 0.13^a	2.24 ± 0.16^a	2.86 ± 0.10^a	2.45 ± 0.15^a	2.90 ± 0.11^a	2.59 ± 0.00^a
OTA1	2.31 ± 0.24^cd	1.98 ± 0.30^ab	1.59 ± 0.00^c	1.43 ± 0.29^bc	2.31 ± 0.24^b	2.08 ± 0.16^bc	2.45 ± 0.15^bc	2.00 ± 0.01^bcd
OTA2	2.31 ± 0.24^cd	1.95 ± 0.43^ab	1.00 ± 0.00^d	0.75 ± 0.50^d	1.90 ± 0.21^e	1.69 ± 0.21^d	1.98 ± 0.30^de	1.44 ± 0.29^f
Silymarin (SL)	2.80 ± 0.17^ab	2.37 ± 0.28^a	2.52 ± 0.13^a	1.90 ± 0.21^abc	2.85 ± 0.20^a	2.45 ± 0.15^a	2.85 ± 0.20^a	2.31 ± 0.24^ab
Vitamin E (VE)	2.99 ± 0.15^a	2.58 ± 0.20^a	2.51 ± 0.23^a	2.06 ± 0.35^ab	2.85 ± 0.20^a	2.45 ± 0.15^a	2.70 ± 0.13^ab	2.23 ± 0.28^abc
OTA1+SL	2.80 ± 0.17^ab	2.39 ± 0.13^a	2.68 ± 0.29^a	2.23 ± 0.28^a	2.85 ± 0.20^a	2.37 ± 0.28^ab	2.80 ± 0.17^a	2.37 ± 0.28^ab
OTA2+SL	1.65 ± 0.47^e	1.04 ± 0.75^c	1.79 ± 0.24^bc	1.69 ± 0.21^bc	2.31 ± 0.24^b	2.08 ± 0.16^bc	2.24 ± 0.28^cd	1.87 ± 0.36^cde
OTA1+VE	2.52 ± 0.13^bc	2.08 ± 0.16^a	2.59 ± 0.01^a	2.16 ± 0.19^a	2.85 ± 0.20^a	2.31 ± 0.24^abc	2.74 ± 0.20^ab	2.39 ± 0.13^ab
OTA2+VE	1.98 ± 0.30^de	1.44 ± 0.29^bc	1.69 ± 0.21^bc	1.40 ± 0.49^c	2.31 ± 0.24^b	1.98 ± 0.30^cd	2.08 ± 0.16^de	1.79 ± 0.24^def
VE+SL	2.67 ± 0.13^abc	2.23 ± 0.28^a	2.58 ± 0.20^a	2.12 ± 0.43^a	2.80 ± 0.17^a	2.31 ± 0.24^abc	2.64 ± 0.11^ab	2.08 ± 0.16^bcd
OTA1+SL+VE	2.80 ± 0.17^ab	2.39 ± 0.13^a	2.75 ± 0.11^a	2.23 ± 0.28^a	2.86 ± 0.10^a	2.51 ± 0.23^a	2.80 ± 0.17^a	2.37 ± 0.28^ab
OTA2+SL+VE	1.65 ± 0.47^e	1.04 ± 0.75^e	1.90 ± 0.21^b	1.44 ± 0.29^bc	2.24 ± 0.16^b	1.98 ± 0.30^cd	1.90 ± 0.21^e	1.54 ± 0.41^ef

Values shown are mean (± SD) from n = 5 birds/group at each timepoint.

Values in each column followed by different letters are significantly different at p ≤ 0.05.

OTA, Ochratoxin A; SL, Silymarin; VE, Vitamin E. All other designations are as outlined in footer of Table 1.

At 14 days post-primary injection, total Ig titers in birds in the OTA1, OTA2, OTA2+VE, OTA2+SL, and OTA2+VE+ SL groups were still significantly lower than in control birds. SL, VE, SL+VE, OTA1+VE, OTA1+SL, and OTA1+SL+VE birds displayed no significant difference in total titers from controls. With respect to IgG, titers found with birds in the SL, VE, SL+VE, OTA1+VE, OTA1+SL+VE, and OTA2+SL groups were not significantly different from controls. In contrast, values for birds in groups OTA1, OTA2, OTA2+VE, and OTA2+VE+SL were significantly lower than the controls. There seemed to be no overall downward trend for OTA1+SL/VE.

At 7 days after the booster SRBC injection, total Ig and IgG titers in birds in groups VE, SL, SL+VE, OTA1+VE, OTA1+SL, and OTA1+VE +SL were not significantly different from control values. At 14 days after the booster injection, total Ig and IgG titers in birds in groups VE, SL, OTA1+SL, OTA1+VE, and OTA1+SL+VE were not significantly different from the control. At each timepoint, in contrast, birds in the OTA1, OTA2, OTA2+VE, OTA2+SL, and OTA2+SL+VE groups had significantly lower titers than the controls.

Lymphoproliferative response to avian tuberculin

Lymphoproliferative responses to avian tuberculin in chicks fed different levels of OTA with or without SL and VE are presented in Table 3. At 24 h post-tuberculin injection, response of chicks in groups SL, VE, SL+VE, OTA1+SL, and OTA1+ SL+VE showed no significant difference from the control values. All other groups had significantly lower values than the controls. At 48 and 72 h post-inoculation, no significant difference in lymphoproliferative responses were noted in any of the test groups.

Table 3.  Lymphoproliferative response (mm) to subcutaneous injection of avian tuberculin.

Groups	24 h post-injection	48 h post-injection	72 h post-injection
Control	0.14 ± 0.03^a	0.10 ± 0.07	0.03 ± 0.03
OTA1	0.06 ± 0.04^bc	0.09 ± 0.07	0.08 ± 0.07
OTA2	0.05 ± 0.02^bc	0.06 ± 0.05	0.09 ± 0.08
Silymarin (SL)	0.11 ± 0.04^ab	0.03 ± 0.03	0.08 ± 0.05
Vitamin E (VE)	0.12 ± 0.03^a	0.09 ± 0.07	0.07 ± 0.04
OTA1+SL	0.14 ± 0.05^a	0.057 ± 0.02	0.06 ± 0.01
OTA2+SL	0.07 ± 0.03^bc	0.03 ± 0.04	0.07 ± 0.06
OTA1+VE	0.02 ± 0.01^c	0.05 ± 0.04	0.08 ± 0.03
OTA2+VE	0.03 ± 0.01^c	0.04 ± 0.03	0.05 ± 0.04
VE+SL	0.14 ± 0.03^a	0.04 ± 0.05	0.07 ± 0.02
OTA1+SL+VE	0.09 ± 0.04^ab	0.03 ± 0.03	0.02 ± 0.02
OTA2+SL+VE	0.05 ± 0.05^bc	0.09 ± 0.07	0.07 ± 0.07

Values shown are mean (± SD) from n = 5 birds/group at each timepoint.

Values in each column followed by different letters are significantly different at p ≤ 0.05.

OTA, Ochratoxin A; SL, Silymarin; VE, Vitamin E. All other designations are as outlined in footer of Table 1.

Mononuclear phagocytic system function assay (carbon clearance assay)

Phagocytic indices of the in situ mononuclear phagocytic system (assessed via carbon clearance) of the treated birds are shown in Table 4. At 3 min post-injection, the phagocytic indices for birds in the SL, VE, SL+VE, OTA1+VE, OTA2+VE, OTA1+SL, and OTA1+ SL+VE groups did not significantly differ from control values. All the other groups’ values were not significantly different from the controls. At 15 min, SL, VE, OTA1 +VE, OTA1+ SL, SL+VE, and OTA1+SL+VE birds displayed no significant differences in indices from the controls.

Table 4.  Phagocytic index as determined by carbon clearance assay.

Groups	At 3 min	At 15 min
Control	0.45 ± 0.08^ab	0.06 ± 0.03^a
OTA1	0.30 ± 0.06^c	0.04 ± 0.01^c
OTA2	0.30 ± 0.06^c	0.03 ± 0.02^cd
Silymarin (SL)	0.44 ± 0.02^ab	0.05 ± 0.01^ab
Vitamin E (VE)	0.41 ± 0.06^ab	0.07 ± 0.02^a
OTA1+SL	0.24 ± 0.06^bc	0.05 ± 0.02^ab
OTA2+SL	0.13 ± 0.08^c	0.02 ± 0.02^de
OTA1+VE	0.61 ± 0.26^a	0.06 ± 0.01^a
OTA2+VE	0.26 ± 0.15^bc	0.02 ± 0.03^de
VE+SL	0.38 ± 0.05^b	0.06 ± 0.01^ab
OTA1+SL+VE	0.34 ± 0.01^bc	0.05 ± 0.01^ab
OTA2+SL+VE	0.11 ± 0.03^c	0.01 ± 0.01^ef

Values shown are mean (± SD) from n = 5 birds/group at each timepoint.

Values were determined as K = (log_n OD₁ − log_n OD₂)/(T₂ − T₁), where OD₁ and OD₂ are optical densities (640 nm) at times T₁ and T₂, respectively. Absorbance from a ‘0 min’ sample was considered as the ‘zero’ value for each respective sample.

Values in each column followed by different letters are significantly different at p ≤0.05.

OTA, Ochratoxin A; SL, Silymarin; VE, Vitamin E. All other designations are as outlined in footer of Table 1.

Discussion

In the present study, feeding SL or VE alone to the birds did not significantly alter results of different immune system end-points as compared against values seen with control birds. Thus, as expected, these agents alone were not immunomodulatory. On the other hand, administration of OTA at both levels resulted in significantly lower values for these same end-points than in the control birds, suggesting they imparted their expected immunosuppressive effects. Of note here, co-administration of SL and/or VE along with 1 or 2 mg/kg OTA was found to have somewhat of an ameliorative effect; however, this classification was made if and only if the values for the end-points in the birds in such groups were not significantly different from those of the control birds.

Antibody responses to SRBC were significantly lower in birds fed OTA alone after both primary and booster doses. Similar results of decreases in humoral immune responses induced by OTA have been reported in broiler chicks (Campbell et al., 1983; Singh et al., 1990; Elkady, 1993; Verma et al., 2004; Elauorssi et al., 2006; Hassan et al., 2010). Decreases in antibody responses to Newcastle Disease vaccine in broiler chicks fed OTA have also been reported by Stoev et al. (2002) and Santin et al. (2002). In the current study, feeding SL or VE to the birds did not significantly alter their antibody responses to SRBC. Thus, as expected, these agents alone were not immunomodulatory. Feeding of SL and VE along with 1.0 mg OTA/kg resulted in non-significant changes in antibody response compared with the control group, suggesting that SL and VE had ameliorated the immunotoxic effects induced by 1 mg/kg OTA. However, a similar ameliorative effect of SL or VE was not observed in chicks fed a higher OTA dose (i.e. 2 mg/kg). Comparative studies dealing with amelioration of OTA effects on humoral antibody responses in chickens—as determined by SRBC antibody titer—are not available in the literature.

Cutaneous lymphoproliferative responses to avian tuberculin in OTA-fed groups were significantly lower than in the control birds, suggesting a decreased T-cell response or a delayed cell-mediated immune (CMI) response. Similar delayed CMI responses to avian tuberculosis have been reported in turkeys fed OTA (Dwivedi and Burns, 1985). A significant decreased in T-cell response to phytohemagglutinin (PHA)-P was noted in chicks produced from the eggs of OTA-fed hens (ul-Hassan et al., 2011a) and in chicks hatched from OTA-injected eggs (ul-Hassan et al., 2011b). Several other reports have presented results similar to those here by using PHA-P instead of avian tuberculin during ochratoxicosis (Elkady, 1993; Santin et al., 2002; Stoev et al., 2002; Verma et al., 2004; Wang et al., 2009). The delayed T-cell response was not significantly different from control birds in chicks given VE and SL (alone or in combination) along with 1 mg OTA/kg, suggesting—for the first time in the literature—that there was an ameliorating effect from both VE and SL on OTA-induced immunotoxicity. On contrast, no amelioration was observed in birds fed VE and SL along with 2 mg OTA/kg.

A lower phagocytic index was noticed here in the OTA-fed chicks. This observation was in accordance with that of Dwivedi and Burns (1984), who reported a decreased index in OTA-treated chicks by using a phenol red clearance test. Li et al. (1999) also reported similar results in fumonisin-fed broiler chicks. The feeding of SL and VE (alone or in combination) along with 1 mg OTA/kg improved the phagocytic index, once again suggesting ameliorative activity of these substances on OTA-induced immunotoxicity. Again, feeding of SL and VE with 2 mg OTA/kg did not result in any improvement in phagocytic index, suggesting no amelioration. Comparative information regarding ameliorative effects of SL/VE on phagocytic activity during ochratoxincosis is not available in the literature.

The exact mechanism by which silymarin ameliorated the immunosuppression in OTA-fed chicks is not well understood. Studies conducted in rats and mice confirmed its non-specific immunostimulating effects. Silymarin was shown to protect skin from injuries due to UVB radiation by reducing the UVB-induced enhanced presence of interleukin (IL)-10 in the skin, altering lymph drainage, and by enhancing the immunostimulatory effects of IL-12 (Meeran et al., 2006). Ajidoost et al. (2006) reported that co-incubation of silymarin with peripheral blood mononuclear cells (PBMC) form of β-thalassemia major patients restored the GSH levels and growth response of these cells towards PHA-P mitogen. Murine lymphocytic proliferation tests employing Concanavalin A (ConA) as well as mixed-lymphocyte cultures (MLC) showed that milk thistle extract (i.e., silymarin) was immunostimulatory (Wilasrusmee et al. 2002a); in these studies, the presence of the silymarin led to increased growth of lymphocytes in the MLC as well as heightened proliferative responses in the mitogen assay. The same investigators (Wilasrusmee et al., 2002b) also used in vitro lymphocyte proliferation assays that measured responses to PHA-P, MLC assays, and assessments of IL-10 and -2 production in the MLC to evaluate the effects of different herbal products on transplant-related immune functions. Ginseng, dong quai, and milk thistle were found to have non-specific immunostimulatory effects. Increase in allo-responsiveness in the MLC assays occurred in the presence of milk thistle and dong quai. It seems that immunostimulatory effects of silymarin similar to those reported in the above-mentioned in vitro/in vivo (rodent) studies might have also played a role in the amelioration of OTA-induced immunotoxic effects in the birds in the present study. However, ongoing/future studies will determine if this is the case or not.

How VE is imparting a protective effect against OTA-induced immunotoxicity is based, in great part, on its anti-oxidant nature. However, VE is also known to affect macrophage functionality and humoral immunity in birds (and hatchlings) by as-yet undefined mechanisms. For example, Gore and Qureshi (1997) reported enhanced macrophages responses after in ovo exposure to VE; these effects were seen to be associated with an improved post-hatch quality of chicks and poults. Further, dietary supplementation with VE or direct injection into eggs resulted in higher antibody responses to killed Newcastle Disease vaccine (Hossain et al., 1998).

In the context of an anti-oxidant, VE has an ability to neutralize lipid peroxidation caused by different toxins, including OTA. Hoehler and Marquardt (1996) reported that OTA in the feed of White Leghorn cockerels resulted in increased malondialdehyde (MDA) levels in their livers. These same authors also noted that supplementation with VE partially ameliorated the peroxidative effects of OTA and led to decreased MDA levels. VE along with other anti-oxidants (such as Vitamin C, selenium, etc.) were shown to ameliorate OTA-induced hepatic apoptosis in mice by enhancing their hepatic anti-oxidant/detoxification systems (Atroshi et al. 2000). In vitro studies examining the anti-oxidant effects of VE against toxicities induced by mycotoxins have also been performed. Baldi et al. (2004) reported that oxidative stress was an important factor in OTA cytotoxicity and that VE (as well as retinol) counteracted OTA-induced oxidative stress.

In conclusion, the results of the present study suggested that both SL and VE possessed the ability to ameliorate the OTA-induced immunotoxic effects in chicks. This amelioration was, however, significant in chicks fed 1 mg OTA/kg and not in those fed 2 mg OTA/kg. Future studies with higher amounts of SL and/or VE will be needed to determine if some SL:OTA or VE:OTA ratios can be established that assure mitigation of OTA-induced immunotoxicities.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.