Immunological status of the progeny of breeder hens kept on ochratoxin A (OTA)-contaminated feed

Abstract

This study aimed to evaluate the immunological status of the progeny of breeder hens kept on ochratoxin A (OTA)-contaminated feed. For this purpose, 84 White Leghorn (WL) layer breeder hens (40-weeks-of-age) were divided into seven groups (A–G). Hens in the Group A were fed a commercial layer ration while those in Groups B–G were kept on a diet amended with 0.1, 0.5, 1.0, 3.0, 5.0, or 10.0 mg OTA/kg, respectively, for 3 weeks. Fertile eggs were set for hatching on the weekly basis to get the progeny of each week separately. Hatched chicks (n = 10 from each group) were euthanized at Day 14 of age, and their immunological organs weighed and fixed in neutral buffered formalin. An indirect immunoperoxidase method was applied to study the frequency of immunoglobulin(s)-bearing cells in the spleen and bursa of Fabricius from these progeny. From other chicks within each set, at Day 16 of age, lymphoblastogenic responses against an intradermal administration of phytohemagglutinin (PHA-P) were determined. Relative weights of the bursa of Fabricius and of the thymus were significantly lower in the progeny of hens fed OTA-contaminated diet for 14 and 21 days. The frequencies of IgA-, IgG-, and IgM-bearing cells were also significantly (P ≤ 0.05) lower in the bursa of Fabricius and spleen of the progeny chicks obtained from dams fed the OTA-mixed diet. Progeny chicks obtained from the breeder hens fed higher doses of OTA showed significantly lower responses to PHA-P than did counterpart chicks from control hens. The findings of this study suggested that there were immunosuppressive effects from OTA in the progeny obtained from breeder hens kept on OTA-contaminated diets.

Keywords::

• Ochratoxin A
• breeder hens
• progeny chicks
• immune system
• immunohistochemistry

Introduction

Fungi are ubiquitous in the environment and mycotoxins, the secondary metabolites of certain species of fungi, are unavoidable contaminants of foods and feeds (Devegowda et al., 1998). The most significant mycotoxins in naturally contaminated poultry feed and feed ingredients are ochratoxins, aflatoxins, T-2 toxin, zearalenone, fumonisins, and deoxynivalenol (Hanif et al., 2006). Ochratoxins include a group of structurally related secondary metabolites produced by several species of genus Aspergillus and Penicillium (Varga et al., 1996; Vega et al., 2006). Aspergillus ochraceus, from which the toxins acquired their name, appears to be the predominant ochratoxin producer (Trenk et al., 1971). Presence of ochratoxin A (OTA), the most toxic among ochratoxins, in the poultry feed and feed ingredients has been reported throughout the globe, including Pakistan (Hanif et al., 2006; Saleemi et al., 2009). The primary effect of OTA is nephrotoxicity, although its immunotoxic, teratogenic, hepatotoxic, neurotoxic, genotoxic, and mutagenic effects have been well reported in various avian and mammalian species (Gilani et al., 1978; Kumar et al., 2004; Elaroussi et al., 2006).

Residues of OTA are detected in tissues such as liver, kidneys, muscles, eggs, and plasma of OTA-fed poultry birds (Frye and Chu, 1978; Micco et al., 1987; Niemiec et al., 1994; Biro et al., 2002). The transfer of OTA in the fertile hen’s eggs leads to decrease in production performance of the hatching chicks (Choudhury et al., 1971). Feeding of OTA-contaminated diet to broiler chicks resulted in decreased weights of immunological organs (Dwivedi and Burns, 1985) and suppression in their humoral and cell-mediated immune responses (Verma et al., 2004; Elaroussi et al., 2006). In an experimental study, Dwivedi and Burns (1984) found a decrease in immunoglobulin-bearing cells in the immunological organs and sera of chicks fed OTA at 2 and 4 mg/kg feed, for 15 days. In that particular study, the OTA-mixed feed was offered for 20 days, with an average feed intake of 80 g/chick/day in the last week of the experiment. This meant that each bird consumed 160 and 320 µg of OTA daily at the 2 and 4 mg OTA levels, respectively, during the last week of experiment. Harvey et al. (1987) reported a similar finding in the bursa of Fabricius of chicks hatched from eggs inoculated with OTA. The immunotoxic effects of OTA are mainly characterized by a reduction in the size/mass of immune system organs such as the spleen, bursa of Fabricius, thymus, lymph nodes, as well as alterations in the function and/or number of immune cells, a depression in host antibody responses, and modulation of the production of cytokines (Al-Anati and Petzinger, 2006).

A lot of information is available about the immunotoxicological effects of direct administration of OTA in mammalian and avian species. However, little is known about the progeny performance of the dams kept on a mycotoxin-contaminated diet. With this in mind, this study aimed to investigate the immunological status of the progeny chicks obtained from breeder hens kept on OTA-contaminated feed.

Materials and methods

Ochratoxin and feed

OTA was produced from A. ochraceus (CECT 2948, Culture Collection Center, University De Valencia, Valencia, Spain) by culturing on wheat grain using a modified method of Trenk et al. (1971). Briefly, 80 g wheat grains were soaked in 80 ml distilled water in a 1000 ml Erlenmeyer flask for 2 h and then autoclaved for 20 min at 121°C. An inoculum prepared from a 2-week-old slant culture of A. ochraceus was mixed with the autoclaved wheat in the flask. The flask was then incubated for 14 days at 28°C in the dark and shaken once daily to break the mycelial growth. On Day 15, the flask was autoclaved at 121°C for 3 min to destroy the mycelial growth. OTA was then extracted from the fermented wheat in acetonitrile-water and quantified using high-performance liquid chromatography (HPLC) and fluorescent detection methods (Bayman et al., 2002).

Basal feed (16% total protein and 2900 Kcal/kg metabolizable energy) was prepared without addition of toxin binder, vitamins, minerals, and antibiotics. Prior to use, each batch of the basal feed was analyzed for ochratoxin, aflatoxin, and zearalenone to ensure that the levels of each were < 1.0 µg/kg. OTA-contaminated feeds were prepared by incorporation of known quantities of OTA. For this purpose, fermented wheat grains were extracted by soaking in a 3-fold quantity of chloroform (1:3) overnight and then filtered through cotton cloth. All the chloroform was then evaporated and the concentrated residues re-suspended into polyethylene glycol (PEG). This suspension was then evenly mixed in the required quantity of basal feed to prepare the experimental feeds containing each desired concentration of OTA. Prior to being used for feeding, the OTA concentration in each experimental diet was verified by HPLC.

Induction of ochratoxicosis in White Leghorn breeder hens

All animal experiments were conducted according to the rules and regulations of the Animal Ethics Committee, Faculty of Veterinary Science, at the University of Agriculture, Faisalabad (Pakistan). Eighty-four White Leghorn (WL) layer breeder hens (40 weeks of age) were procured from a breeder farm and then kept in battery cages in the Department of Pathology at the University of Agriculture Faisalabad, Pakistan. After acclimatization for 1 week on basal layer ration, birds were divided into seven groups (A–G), each consisting of 12 birds. Hens in Group A were kept on basal layer ration, while those in Groups B, C, D, E, F, and G were fed OTA-contaminated feed at 0.1, 0.5, 1.0, 3.0, 5.0, or 10.0 mg OTA/kg feed, respectively, for a period of up to 21 days. In this study, there was an average feed intake of ≈105 g/hen/day over the course of the experiment. This meant that each bird consumed from 10.5 to 1050 µg OTA daily (at the corresponding range from the lowest to highest OTA levels, respectively) during the 21-day period.

Healthy breeder roosters of WL were maintained on basal layer ration for semen collection. Semen was collected by an abdominal massage method and diluted (1:1) with semen extender (OVODYL, IMV, L’Aigle Cedex, France). Laying hens were inseminated on alternate days by using artificial insemination gun and straws (IMV). Hatching eggs were then collected on a daily basis and set for incubation on a weekly basis. Prior to setting, eggs were stored at 15°C. The progeny, which were obtained on a weekly basis, were maintained separately for 4 weeks under standard environmental conditions, i.e., on basal layer ration verified not to contain OTA or aflatoxin-B₁ (AFB₁) residues at a level > 1.0 µg/kg ration.

Necropsy and sample collection

On Day 14, 10 chicks from each group were euthanized and their bursa of Fabricius, spleen, and thymus were harvested. These organs were weighed and bursa of Fabricius and spleen were then fixed in 10% neutral buffered formalin in order to permit a counting of immunoglobulin-bearing cells via the method described by Khan et al. (1997).

Immunohistochemical methods

Tissues fixed in the buffered formalin were embedded in paraffin following conventional histopathological procedure. Sections (5-µm thickness) were then cut using a rotary microtome and placed on glass slides. To perform an indirect immunoperoxidase-staining of the samples, normal rabbit serum (cat. no. 16101, lot no. 455567A, Bethyl Labs, Montgomery, TX) was diluted (1:200) with 0.01 M phosphate-buffered saline (PBS, pH 7.4). Goat anti-chicken-IgG (cat. no. A30-106A), -IgM (cat. no. A30-102A), and -IgA (cat. no. A30-103A) were each diluted to 1:800 in PBS. For detection, secondary horseradish peroxidase (HRP)-conjugated rabbit anti-goat IgG (cat. no. A50-100P) was diluted to 1:500 in PBS.

After de-waxing with xylene, tissues on each slide were fixed in methanol for 30 min at room temperature (RT). Endogenous peroxidases in each sample were inhibited with 0.3% H₂O₂ (Merck, Darmstadt, Germany) in PBS for 30 min at RT. After washing H₂O₂ from the tissue section with PBS, the section was covered with diluted normal rabbit serum and incubated for 1 h in a moist chamber at RT. Thereafter, residual serum was drained off and the slide washed with PBS; the appropriate primary antibody (IgG, IgA, or IgM) was then applied on the slide and incubated for 18 h at 4°C in a moist chamber. Any unbound primary antibody was then rinsed away by washing with PBS for 5 min at RT. The secondary antibody was then placed on the section and incubated for 1 h in a moist chamber at RT. Unbound antibodies were then removed by washing in PBS for 5 min at RT; the washing steps were repeated three times and any excess PBS was then wiped from the slide. For color development, DAB solution (3,3’-diaminobenzidine, cat. no. TA-060-HDX, Thermo Scientific, Rockford, IL) was prepared by combining 40 µl DAB plus a manufacturer-supplied chromogen (TA-002-HCX) with 2 ml of DAB plus manufacturer-supplied substrate (TA-060-HSX). DAB solution was applied on the section and incubated for 10 min in a moist chamber at RT. The DAB solution was then washed from the sections using PBS for 5 min at RT. After thorough washing, the section was counterstained in hematoxylin solution for 30 sec. An excess hematoxylin solution was removed by washing under tap water for 10 min. A dehydration and clearing process was then performed through a series of ascending grades of alcohol and two batches of xylene, following conventional histopathological procedure. Finally, the section was cover-slipped using DPX mountant (Merck-BDH, Lutterworth, England).

During histoplanimetry under an M7000DB light microscope (Swift, Tokyo, Japan), the immunoglobulin(s)-bearing cells were counted in three sections of spleen and bursa/bird. From each section of bursa of Fabricius, 20 microscopic fields were selected for counting from the modullary areas of randomly selected follicles. With each spleen, plasma cells were counted in 20 randomly selected fields/section, at 400× magnification using an ocular micrometer, and their relative frequency/0.1 mm² was calculated (Weibel, 1969; Khan et al., 1997).

Lymphoproliferative response to phytohemagglutinin

At Day 16 of age, 12 birds from each group were injected with phytohemagglutinin (PHA-P) (MP Biomedicals, Inc., Cleveland, OH) to study the lymphoblastogenic response described by Corrier (1990). For this, 100 µg PHA-P (dissolved in 0.1 ml normal saline) was injected into the intradigital space between digits 3 and 4 of the right foot. At the same time, 0.1 ml normal saline was injected into a similar site in the left foot. Skin thickness was then measured 24 and 48 h post-injection using a constant tension micrometer (Global Sources, Shanghai, China). The thickness response was then calculated as: cutaneous basophilic hypersensitivity response = (right foot skin thickness − left foot skin thickness) at each timepoint.

Statistical analysis

All data were subjected to analysis of variance tests. Means of the different groups were compared using a Duncan’s Multiple Range test within a MSTATC statistical package (Department of Crop and Soil Sciences, Michigan State University, East Lansing, MI). Data were considered significantly different from one another at a P-value ≤ 0.05.

Results

Relative weights of immunological organs

The relative splenic weights in the progeny of hens fed OTA-contaminated diet were not significantly affected (as compared to values seen in control hen progeny) regardless of the length of the dietary exposures (Table 1). In contrast, the progeny of breeder hens fed OTA contaminated diet at higher doses (i.e., Groups E, F, and G) for 14 and 21 days showed significantly lower relative bursa of Fabricius weights compared to values in control progeny counterparts. With the longest exposure (i.e., for 21 days), the differences among the progeny of hens in these groups reflected significant dose-dependencies. Finally, there were no significant differences (as compared to control hen progeny) in the relative thymus weights of hens fed any of the OTA-contaminated diet for 7 and 14 days; however, when hens were fed any of these diets for 21 days, the progeny all expressed significantly lower weights. Unlike what was noted with the bursa results, there were no dose-dependencies observed with this organ.

Table 1.  Relative organ weights (% of body weight) of progeny chicks at the age of 2 weeks.

Group (mg OTA/kg)	A (0)	B (0.1)	C (0.5)	D (1.0)	E (3.0)	F (5.0)	G (10.0)
Progeny obtained from hens fed OTA-bearing feed for 7 days
Bursa of Fabricius	0.28 ± 0.04	0.27 ± 0.12	0.29 ± 0.03	0.27 ± 0.07	0.28 ± 0.05	0.22 ± 0.03	0.28 ± 0.04
Spleen	0.12 ± 0.04	0.15 ± 0.08	0.11 ± 0.01	0.12 ± 0.03	0.13 ± 0.03	0.10 ± 0.04	0.12 ± 0.04
Thymus	0.35 ± 0.14	0.27 ± 0.11	0.31 ± 0.06	0.39 ± 0.14	0.31 ± 0.17	0.47 ± 0.20	0.35 ± 0.14
Progeny obtained from hens fed OTA-bearing feed for 14 days
Bursa of Fabricius	0.31 ± 0.02^a	0.29 ± 0.08^a	0.31 ± 0.07^a	0.27 ± 0.09^a	0.23 ± 0.07^ab	0.15 ± 0.02^b	0.14 ± 0.02^b
Spleen	0.11 ± 0.04	0.10 ± 0.04	0.13 ± 0.03	0.09 ± 0.04	0.11 ± 0.04	0.12 ± 0.06	0.08 ± 0.01
Thymus	0.20 ± 0.02	0.22 ± 0.05	0.17 ± 0.03	0.15 ± 0.02	0.16 ± 0.05	0.16 ± 0.01	0.15 ± 0.02
Progeny obtained from hens fed OTA-bearing feed for 21 days
Bursa of Fabricius	0.28 ± 0.02^a	0.26 ± 0.01^b	0.25 ± 0.01^c	0.21 ± 0.02^e	0.22 ± 0.01^d	0.17 ± 0.02^f	0.15 ± 0.03^g
Spleen	0.26 ± 0.03	0.22 ± 0.04	0.23 ± 0.05	0.24 ± 0.05	0.23 ± 0.07	0.23 ± 0.06	0.16 ± 0.01
Thymus	0.38 ± 0.09^a	0.20 ± 0.07^b	0.24 ± 0.10^b	0.14 ± 0.06^b	0.16 ± 0.10^b	0.19 ± 0.08^b	0.22 ± 0.05^b

Values (mean ± SD) in each row (within a given set of progeny) followed by different letters are significantly different (P ≤ 0.05) from one another.

Frequency of immunoglobulin-bearing cells in the spleen and bursa of Fabricius of progeny chicks

Scoring of immunoglobulin-bearing plasma cells/0.1 mm² in the spleen and bursa of Fabricius of progeny of hens fed OTA is presented in Table 2. Following exposure to OTA-contaminated diets for 7 days, the numbers of IgA- and IgM-bearing plasma cells in the spleens of progeny from hens in Groups D, E, F, and G were significantly lower than in samples from chicks of Group A hens (controls). In contrast, the numbers of IgG-bearing cells were not seen to differ significantly among any of the various progeny. In the bursa of Fabricius, the numbers of IgA-bearing cells were significantly lower in all the groups obtained from the OTA-fed dams compared with levels noted with progeny of hens in Group A (representative immunostained sample provided in Figure 1). This decrease in the number of cells was seen to be dose related. In the case of IgG, significantly lower numbers of cells were found in samples of progeny from hens in Groups E, F, and G as compared with samples from Group A hen chicks. Finally, the numbers of IgM-bearing cells were also seen to be significantly lower in samples from progeny of Groups D, E, F, and G hens as compared with those from their control progeny counterparts.

Table 2.  Immunohistochemical scoring (specific type of Ig-bearing cell/0.1 mm²) in the spleen and bursa of Fabricius of progeny of hens fed OTA-contaminated feed.

Group (mg OTA/kg)	Spleen			Bursa
	IgA	IgG	IgM	IgA	IgG	IgM
Progeny obtained from hens fed OTA-bearing feed for 7 days
A (0)	73.2 ± 7.6^a	27.2 ± 4.4	51.4 ± 1.3^a	571.4 ± 5.9^a	509.0 ± 17.6^a	56.6 ± 10.3^a
B (0.1)	70.0 ± 1.6^a	25.9 ± 1.6	46.5 ± 2.9^ab	537. ± 3.7^b	524.3 ± 18.7^a	49.4 ± 2.2^ab
C (0.5)	69.8 ± 6.7^a	25.9 ± 1.9	48.2 ± 3.8^ab	509.3 ± 15.5^c	532.7 ± 14.8^a	49.8 ± 4.4^ab
D (1.0)	56.2 ± 1.2^b	23.5 ± 4.3	44.2 ± 5.0^bc	413.0 ± 8.3^d	530.0 ± 17.4^a	44.8 ± 2.8^b
E (3.0)	44.4 ± 5.9^c	24.1 ± 1.6	37.9 ± 2.2^d	417.9 ± 17.2^d	457.8 ± 15.9^b	44.2 ± 0.9^b
F (5.0)	42.6 ± 2.1^c	23.6 ± 3.1	39.9 ± 4.6^cd	377.2 ± 20.7^e	430.9 ± 17.66^b	46.9 ± 1.6^b
G(10.0)	24.1 ± 2.8^d	21.4 ± 1.4	28.8 ± 2.2^e	357.4 ± 25.2^e	234.8 ± 11.7^c	34.8 ± 2.8^c
Progeny obtained from hens fed OTA-bearing feed for 14 days
A (0)	94.6 ± 5.0^a	29.4 ± 10.0^a	63.0 ± 15.0^a	586.6 ± 8.4^a	537.9 ± 21.6^a	29.4 ± 0.9^a
B (0.1)	99.8 ± 4.8^a	25.1 ± 2.0^ab	59.3 ± 4.9^ab	576.1 ± 9.8^a	556.2 ± 32.2^a	25.3 ± 1.1^b
C (0.5)	91.6 ± 1.6^a	24.7 ± 1.1^ab	53.1 ± 0.6^abc	516.7 ± 15.0^b	533.1 ± 48.1^a	23.7 ± 1.9^bc
D (1.0)	75.9 ± 3.4^b	23.0 ± 3.6^abc	49.0 ± 5.7^bcd	445.7 ± 11.1^c	520.4 ± 15.10^ab	20.8 ± 2.3^c
E (3.0)	72.4 ± 15.9^b	22.4 ± 3.6^abc	42.4 3.4^cd	332.1 ± 13.5^e	481.3 ± 8.0^b	22.2 ± 2.5^bc
F (5.0)	69.3 ± 3.5^b	18.9 ± 4.7^bc	37.2 ± 2.9^d	342.6 ± 14.8^de	421.8 ± 9.6^c	23.7 ± 2.5^bc
G(10.0)	46.3 ± 3.9^c	14.2 ± 2.8^c	38.9 ± 7.1^d	363.2 ± 23.4^d	362.6 ± 29.1^d	17.3 ± 1.6^d
Progeny obtained after 21 days of OTA feeding to hens
A (0)	77.0 ± 1.6^a	41.8 ± 4.0^a	53.7 ± 10.5^a	555.6 ± 7.9^a	610.7 ± 15.9^a	45.3 ± 1.0^a
B (0.1)	70.4 ± 1.1^ab	38.7 ± 0.4^ab	45.1 ± 2.5^ab	538.1 ± 11.4^a	627.6 ± 2.5^a	47.3 ± 2.2^a
C (0.5)	60.1 ± 7.7^bc	35.2 ± 8.3^abd	34.0 ± 3.8^cd	559.2 ± 22.6^a	614.4 ± 14.7^a	42.6 ± 2.2^ab
D (1.0)	56.6 ± 4.6^c	32.5 ± 6.0^bcd	39.7 ± 6.2^bc	474.9 ± 30.8^b	607.4 ± 19.0^a	39.9 ± 1.6^bc
E (3.0)	54.9 ± 3.4^c	27.6 ± 5.0^cd	34.8 ± 2.8^bcd	464.4 ± 12.5^b	556.2 ± 4.3^b	39.7 ± 1.4^c
F (5.0)	56.0 ± 7.4^c	24.9 ± 2.2^d	38.7 ± 5.0^bc	451.4 ± 20.0^b	544.8 ± 9.8^b	36.8 ± 0.7^d
G(10.0)	36.0 ± 10.5^d	26.1 ± 2.9^d	26.1 ± 2.9^d	324.5 ± 9.4c	551.4 ± 11.0^b	39.1 ± 0.7^cd

Values (mean ± SD) in each column (within a given set of progeny) followed by different letters are significantly different (P ≤ 0.05) from one another.

Figure 1.  Photomicrograph of bursa of Fabricius from representative progeny chick of a Group A hen (control) fed OTA-free feed. IgG-bearing cells in medullary region shown here display positive DAB staining (DAB & Hematoxylin stain; 100× magnification).

Following exposure to OTA-contaminated diets for 14 days, the number of IgA- and IgM-bearing plasma cells in the spleens of progeny from hens in Groups D, E, F, and G was significantly lower than in samples from chicks of Group A hens (controls). In the case of IgG-bearing cells, significantly lower numbers of plasma cells were only found in progeny from hens in Groups F and G. In the bursa of Fabricius, the numbers of IgA-bearing cells were significantly lower (relative to levels in control progeny tissues) in all the progeny groups except those from hens in Group B (representative photomicrograph shown in Figure 2). The numbers of IgM-bearing cells were significantly lower in all groups compared with the controls. With respect to IgG-bearing cells, there were only significantly lower numbers (compared with those in organs from chicks born of hens in Group A) observed in tissues recovered from progeny from hens in Groups E, F, and G.

Figure 2.  Photomicrograph of bursa of Fabricius from representative progeny chick of a Group B hen fed OTA-contaminated feed at 0.1 mg/kg for 14 days. IgA-bearing cells seen here display positive DAB staining (DAB & Hematoxylin stain; 100× magnification).

Following exposure to OTA-bearing diets for the longest period (i.e., 21 days), the numbers of IgA- and IgM-bearing plasma cells in the spleens were significantly lower (relative to levels in control progeny tissues) in all the progeny groups except those from hens in Group B. In the case of IgG-bearing cells, the number of plasma cells was significantly lower (relative to that in control counterparts) in the organs obtained from progeny of hens in Groups D, E, F, and G. In the bursa of Fabricius, the numbers of IgA- and IgM-bearing cells were significantly lower (relative to those in control progeny tissues) for the progeny of hens in Groups D, E, F, and G (a representative immunostained sample from the last Group is provided in Figure 3) compared with Group A. In case of IgG-bearing cells, significantly lower numbers of plasma cells were noted among progeny of hens from Group E (representative photomicrograph is shown in Figure 4), as well as from Groups F and G, as compared with those in control chick tissues.

Figure 3.  Photomicrograph of bursa of Fabricius from representative progeny chick of a Group G hen fed OTA-contaminated feed at 10 mg OTA/kg feed for 21 days. The relatively few IgA-bearing cells (DAB stained) present here are indicated by arrow heads (DAB & Hematoxylin stain; 600× magnification).

Figure 4.  Photomicrograph of bursa of Fabricius from representative progeny chick of a Group E hen fed OTA-contaminated feed at 3 mg OTA/kg feed for 21 days. IgG-bearing cells in medullary region shown here display positive DAB staining (DAB & Hematoxylin stain; 100× magnification).

Lymphoblastogenic responses to PHA-P

Lymphoproliferative responses to intradermal administration of PHA-P into the toe web of the chicks of hens fed OTA-spiked diets for varying periods of time were assessed (Table 3). Among the progeny obtained from hens fed the OTA-bearing diet for 7 days, there was immunosuppression in the chicks of hens fed the higher doses of OTA (i.e., Groups E, F, and G). At 24 h after the PHA-P injection, the skin thickness response was significantly lower in the progeny of these hens compared with that with progeny from hens in Group A. These differences (in fact, those among all the groups) became, however, non-significant by 48 h postinjection. Among the progeny obtained from hens fed the OTA-bearing diet for 14 days, there were significantly lower responses at both 24 and 48 h post-PHA-P injection in progeny of hens from Groups E, F, and G compared with that with progeny from hens in Group A. Finally, among the progeny obtained from hens fed OTA-spiked feed for 21 days, there were significantly lower responses (compared with that of progeny from hens in Group A) to PHA-P at 24 h only among progeny of hens from Groups F and G. Interestingly, at 48 h post-injection, only the progeny of hens from Group G still evinced the significantly lower responses than control progeny counterparts.

Table 3.  Lymphoblastogenic response against PHA-P in progeny of OTA-fed breeder hens.

Increased skin thickness (mm)	Groups (mg OTA/kg feed)
	A (0)	B (0.1)	C (0.5)	D (1.0)	E (3.0)	F (5.0)	G (10.0)
Progeny obtained from hens fed OTA-bearing feed for 7 days
After 24 h	0.79 ± 0.09^a	0.67 ± 0.03^a	0.66 ± 0.04^a	0.65 ± 0.16^a	0.46 ± 0.02^b	0.45 ± 0.04^b	0.22 ± 0.13^c
After 48 h	0.60 ± 0.21	0.51 ± 0.12	0.38 ± 0.06	0.34 ± 0.34	0.37 ± 0.15	0.26 ± 0.26	0.22 ± 0.13
Progeny obtained from hens fed OTA-bearing feed for 14 days
After 24 h	0.75 ± 0.10^a	0.60 ± 0.09^ab	0.54 ± 0.13^ab	0.47 ± 0.13^ab	0.43 ± 0.11^b	0.36 ± 0.26^b	0.38 ± 0.19^b
After 48 h	0.56 ± 0.13^a	0.53 ± 0.04^a	0.46 ± 0.03^ab	0.46 ± 0.16^ab	0.29 ± 0.11^bc	0.20 ± 0.16^c	0.10 ± 0.05^c
Progeny obtained from hens fed OTA-bearing feed for 21 days
After 24 h	0.71 ± 0.11^a	0.69 ± 0.05^a	0.63 ± 0.10^a	0.58 ± 0.12^ab	0.55 ± 0.05^ab	0.44 ± 0.05^bc	0.32 ± 0.12^c
After 48 h	0.43 ± 0.04^a	0.39 ± 0.15^a	0.39 ± 0.10^a	0.30 ± 0.05^ab	0.29 ± 0.04^ab	0.22 ± 0.18^ab	0.10 ± 0.14^b

Values (mean ± SD) in each row (within a given set of progeny) followed by different letters are significantly different (P ≤ 0.05) from one another.

Discussion

In this study, the relative weights of the bursa of Fabricius and thymus were significantly lower in the progeny of breeder hens kept on OTA-contaminated diet for 14 and 21 days. To our knowledge, there is no report available in the literature describing alterations in immunological organ weights of progeny chicks obtained from dams fed OTA-contaminated feed. However, there has been a study that described a decrease in the body weights of progeny chicks obtained from breeder hens kept on OTA-contaminated rations (Choudhury et al., 1971). A decrease in the weight of immunological organs due to OTA feeding has been reported in broiler chicks (Singh et al., 1990; Stoev et al., 2002). It is possible that the noted decreases in the weights of immunological organs in this study are due to cytotoxic/necrotic effects of OTA residues in the eggs (data not shown) acting, in part, upon the developing organs of the chicks. The basis for this notion is the results of the studies by Qureshi et al. (1998) that reported decreases in the immune status of the progeny of breeder hens fed AFB₁-contaminated diets. Nevertheless, although OTA levels in the hens’ eggs in this study were not analyzed, transfer of the mycotoxin to (and thus the potential for cytotoxic/necrotic effects in) the eggs was likely. This claim can be inferred from the studies by Piskorska-Pliszczynska and Juszkiewicz (1990) that showed that for up to 4 days following administration of single doses of OTA (i.e., 1, 5, or 20 mg OTA/kg body weight), traces of intact mycotoxin could be found in the eggs of exposed Japanese quail hens.

The frequency of IgG-, IgA-, and IgM-bearing cells in the spleen and bursa of Fabricius was significantly lower in the progeny of hens that were maintained on OTA-contaminated feeds. These findings are in keeping with those of earlier studies of various avian/mammalian species exposed to OTA. With respect to avian species, Harvey et al. (1987) reported a decrease in the levels of IgG-bearing cells in the bursa of Fabricius of chicks newly hatched from OTA-inoculated eggs (i.e., 2.5–7.9 µg OTA administered to 13-day-old chicken embryos via the chorioallantoic membrane). Dwivedi and Burns (1984) also saw a similar trend in levels of Ig-bearing cells in the immune system organs and sera of chicks directly fed 2.0 or 4.0 mg OTA/kg feed for 15 days.

This decrease in the number of Ig-bearing cells may be due to direct inhibitory effects of OTA (transferred in the eggs) on protein synthesis as described by Creppy et al. (1983). It is known that a part of the toxicity of OTA is due to a structural homology with the amino acid phenylalanine, resulting in an inhibition of protein synthesis (due to a competition for the specific tRNA; Creppy et al., 1979). Most recently, Stoev (2010) hypothesized these effects on tRNA, apart from being an underlying basis for their earlier observation of OTA effects on vaccinal immunity in exposed (at levels of 130, 305, and 790 µg OTA/kg diet) broiler chicks (Stoev et al., 2000), might also be a basis for the reduced egg production manifest by hens exposed to OTA-contaminated feeds (1–5 mg OTA/kg diet) for up to 1 year. Based on these other studies purporting of this particular mechanism of toxicity, it is likely a measure of OTA-induced impairment of protein synthesis had to have occurred in the exposed hens/chicks here. However, it remains unclear whether the decreases in the number of Ig-bearing cells here were due to changes in the numbers of the cells per se in either organ, in the abilities of the cells present to produce the Ig surface proteins, or both. Further studies are needed to clarify this matter.

Although OTA-induced (direct) effects could have lead to decreased Ig synthesis by the hosts’ B-lymphocytes in situ, the decreases in levels of Ig-bearing cells could also have been a result of apoptotic/necrotic changes induced by OTA in follicular regions of the hosts’ immune system organs. Such an effect from the mycotoxin would have had to exceed the amount of loss of Ig-bearing cells that normally occurs in the chicken bursa of Fabricius, i.e., there is a decrease in surface Ig expression on B-lymphocytes that precedes their death by apoptosis (Paramithiotis et al., 1995).

The changes in the levels of antibody formation/ immunoglobulin-bearing cells have implications for several immune responses in situ. Among these is the response to a mitogen such as PHA-P, which is considered a good in vivo measure of T-lymphocyte function (Qureshi et al., 1997). In this study, feeding of OTA to breeder hens resulted in significant decreases in the skin thickness response to PHA-P in their progeny chicks. This outcome could be the result of alterations in the functions of T-lymphocytes/depression in the mass of thymus (reflective of a likely depression in subsequent numbers of circulating mature lymphocytes to partake in the PHA-P-induced skin thickening response) in the progeny chicks from the OTA-fed breeder hens.

Decreases in the weights of immunological organs, the numbers of antibody-bearing cells, and in skin thickness in response to intradermal PHA-P inoculation suggests that there is a substantive potential for deleterious effects of OTA that might be transferred to eggs and then impact upon the development of the developing chick’s immune system/associated organs. To our knowledge, this study is the first to report on the immunotoxic effects of OTA in the progeny of breeder hens fed OTA-contaminated feed. Nevertheless, we are mindful that to provide fuller support to our conclusions/hypotheses here, our future studies will need to verify that: (a) OTA, at the doses used here (and which we recognize are lower than those used in some of the other studies we reference), transfers from the hens to their eggs (and at what concentrations): and, (b) OTA, at those transferred concentrations (i.e., administered to 13-day-old chicken embryos via the chorioallantoic membrane in the manner used by Harvey et al. [1987]) induces the effects we are observing in the studies we report here.

Declaration of interest

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