Immunopathological effects of experimental T-2 mycotoxicosis in Wistar rats

Abstract

It is well known that T-2 toxin has cytotoxic radiomimetic like effects on the immune system. Because of scant research data demonstrating the chronic effects of low doses of the T-2 toxin on humoral and cellular responses in rats, the present experiment was undertaken. The animals were divided into four groups, namely, group I (0.5 ppm), group II (0.75 ppm) and group III (1.0 ppm) and group IV (control) were given toxin-free diet for 12 weeks and eight animals each were sacrificed at 2, 4, 6, 8, 10, and 12-week of the experimental period. The humoral immune response was evaluated based on hemagglutination test (HA), and levels of serum immunoglobulins (IgA, IgG, IgM) while the cell-mediated immune response was evaluated by delayed-type hypersensitivity (DTH) response to ovalbumin, lymphocyte stimulation index, analyses of CD4⁺ and CD8⁺ T lymphocytes and mRNA expression levels of selected cytokines like IL-2, IFN-γ, IL-4 and IL-10 by quantitative Real-time PCR in experimental groups. T-2 treatment caused suppression in both humoral and cell-mediated immune responses as evidenced by a decrease in all these parameters in toxin fed animals compared to the control in the dose and duration-dependent manner. This dose-dependent effect on the immune system has been further reflected largely by the depletion of lymphocytes from lymphoid organs as observed histopathologically in the spleen, thymus, and Peyer’s patches in the present study.

Keywords

T-2 toxin immune response cytokines expression qRT PCR immunoglobulins assay pathomorphological study Wistar rats

Introduction

Mycotoxins are the secondary metabolites of low molecular weight produced mainly by the molds (filamentous fungi), which grow as pathogens on plants or as saprophytes on stored plants and grains such as wheat, maize, oats, barley, rice, beans, and soya beans as well as in some cereal-based products.¹ The Food and Agricultural Organization (FAO) estimates that approximately 25% of the world’s agricultural commodities are contaminated to some extent with mycotoxins suggesting that mycotoxins are a constant concern.² The consumption of food and feed material contaminated by mycotoxins is a potential health hazard for both humans and animals. Acute and long-term toxicity of mycotoxin may result in teratogenic, carcinogenic, mutagenic, and immune-suppressive effects in animals and birds on the consumption of a mycotoxin contaminated diet.³

The most important trichothecene mycotoxins are produced mainly by species of the genus Fusarium.⁴ The trichothecenes are characterized by the 12, 13-epoxytrichothec-9-ene ring structures, and this 12, 13-epoxy ring is responsible for their toxicological action.⁵ Though more than 200 trichothecenes have been identified to date, but major trichothecenes that have been found to contaminate food or animal feed include T-2 toxin, diacetoxyscirpenol (DAS), deoxynivalenol (DON) and nivalenol (NIV) and some less frequently occurring derivatives of these toxins, such as 3-acetyldeoxynivalenol (3ADON), 15-acetyldeoxynivalenol (15ADON), FUS and HT-2 toxin.

Out of all these toxins, T-2 toxin is one of the most potent cytotoxic mycotoxin, which is known to produce fatal toxic reactions in humans and animals.⁶ T-2 toxin (3, hydroxy-4, 8, 15-diacetoxy-8a-(3-methylbutyryloxy)-12, 13epoxytrichothec-9-ene) was first isolated from the mold Fusarium tricinctum (Fusarium sporotrichioides).⁷ T-2 toxin is a non-volatile, low-molecular-weight (MW 466.52) compound, insoluble in water and petroleum ether, but readily soluble in chemicals such as acetone, ethyl-acetate, ethyl alcohol, methyl alcohol, chloroform, dimethyl sulfoxide, and propylene glycol. It is highly resistant to inactivation by heat and UV light. Therefore, it is not commonly inactivated during the routine process of food production or by autoclaving.

Toxicodynamic studies demonstrated the mechanism of action of T-2 toxicity that it inhibits DNA, RNA, and protein synthesis in eukaryotic cells, affects the cell cycle and induces apoptosis both in vivo and in vitro.⁵ Actively dividing cells, such as those lining the gastrointestinal tract, skin, bone marrow, lymph nodes, spleen, and liver were found to be highly sensitive to T-2 toxin resulting in leucopenia and consequently increased susceptibility to infection with various pathogens.⁸

Different surveys reported the presence of T-2 toxin in different food grains sampled from Asia, South Africa, South America, North America, Europe, and other countries which ranged from 0 to 10 μg/g with rare exceptions up to the levels of 40 μg/g.⁹ Ramakrishna et al. studied the occurrence of mycotoxins in selected staple feeds in India and observed the presence of T-2 toxin to the rain-affected samples of wheat (0.55–4.0 µg/g) and wheat products such as refined wheat flour (0.8 µg/g).¹⁰ Haubruge et al. reported the incidence of T-2 toxin in grain samples (65% to 76%) with mean concentration from 0.01 mg/kg to 0.1 mg/kg and maximum value of 3.75 mg/kg.¹¹

A large number of experimental studies in different experimental animals have been conducted where they received T-2 toxin as a single high dose level. However, under field conditions, animals mostly receive contaminated food and feed material with a low level of toxins for a long duration of time. To the best of our knowledge, limited investigations have been undertaken to study the chronic toxic effects of T-2 mycotoxin and no detailed systematic work, particularly on chronic exposure to low doses of the T-2 toxin has been undertaken. Therefore, the present study was conducted through a planned experimental design as per standard protocol to elucidate T-2 induced immunopathology in Wistar rats.

Materials and methods

Production of T-2 toxin

A known culture of Fusarium sporotrichioides var. sporotrichioides (MTCC1894), procured from the Institute of Microbial Technology (IMTC), Chandigarh, India, was used to produce the T-2 mycotoxin in the laboratory. The stock culture was subcultured in Sabouraud’s dextrose agar media. T-2 mycotoxin production was done as per the method described by AOAC with some modifications.¹² To prepare the substrate, maize was partially ground and soaked with 40% broth (10% peptone, 40% glucose and distilled water) for 2 hours (h), and intact wheat grains were soaked with 60% distilled water overnight. The grain mixtures were put in flasks (100 g in 500 ml flask, 300 g in 1000 ml flask, 500 g in 2000 ml flask, and 750 g in 3000 ml flask) and sterilized by autoclaving at 15 pound (lbs) pressure for 15 minutes (min.). Loop full inoculum containing freshly grown mycelia was added to each flask under proper sterile conditions and kept in B.O.D incubator at 16 ± 1°C. Flasks were shaken thoroughly twice a day to break clumps and to ensure the uniform spread of fungus during the period of incubation. Cotton white type growth of Fusarium sporotrichioides on wheat appeared in 4–5 days post culture. By the end of 8–10 days light pink hue, color growth of the fungus was observed. After the adequate quantity of fungal growth (3 weeks post-incubation), the cultures in flasks were autoclaved at 15 lbs for 30 min to destroy the mycelia and spores, dried at 80°C for 10 h and ground to a fine powder for toxin analysis.

Quantification of T-2 toxin

The produced T-2 toxin was analyzed and quantified by thin-layer chromatography (TLC) and spectrophotometry at Animal Feed Analytical and Quality Control Laboratory (AFAQCL), Veterinary College and Research Institute, Namakkal, Tamil Nadu (India). It was further confirmed by using an enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (Agra-quant T-2 Toxin Test kit; Romer Labs, USA). The cultured maize and wheat powder containing a known amount of T-2 mycotoxin were kept in a clean and dry container at room temperature.

Preparation of experimental feed and diet

The cultured wheat or maize powder containing known amounts of T-2 toxin was added and thoroughly mixed with the basal ration which was earlier tested negative for the presence of the most commonly occurring mycotoxins such as aflatoxin, ochratoxin, and T-2 toxin in a proportion to obtain the desired concentration of T-2 toxin in the diet i.e. 0.5 ppm (0.5 mg/kg feed), 0.75 ppm (0.75 mg/kg feed) and 1.0 ppm (1 mg/kg feed). Aliquots were taken from this mixed diet and further toxin was quantified by TLC and spectrophotometry and ELISA to ensure proper mixing and the desired concentration in the experimental diet.

Experimental animals

A total of 192 male Wistar rats (21 days of age), weighing 60–80 g were procured from the Laboratory Animal Resources (LAR) section of the Indian Veterinary Research Institute, Izatnagar, Bareilly, India and housed in the Experimental Laboratory Animal shed of the Division of Pathology. Wistar rats were chosen, because of their small size, low cost, and practical convenience in housing and maintenance of large numbers of animals. The rat has been exclusively recommended as the choice of animal species for such toxicological studies.¹³ The rats were maintained under standard management conditions of controlled temperature (22 ± 3°C) and humidity (45 ± 15%) with 12:12 h light to dark cycle and examined for abnormality or overt ill health if any. They were given standard feed and water ad libitum throughout the experimental period. The feed was pre-tested for contamination of commonly occurring mycotoxins (Aflatoxin B₁, Ochratoxin A, and T-2 toxin) and the only toxin-free feed was used to make the experimental diets. The animal experimentation protocol was approved by the Institutional Animal Ethics Committee (IAEC) (vide No. IVRI/PATH/09-12/002) and the experiment on animals was performed based on the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India.

Experimental design

After an acclimatization period of 7 days, all the animals were weighed and randomly assigned to four groups of 48 rats each to give approximately equal initial group mean body weights. Following allocation, the animals were marked with the picric acid solution for individual identification. The experimental animals were given T-2 toxin in feed at different levels i.e. groups I: 0.5 ppm, group II: 0.75 ppm and group III: 1.0 ppm and group IV was served as control and given toxin-free diet for 12 weeks and eight animals from each group were sacrificed at 2, 4, 6, 8, 10, and 12-week intervals. Three doses of T-2 toxin (0.5, 0.75, and 1.0 ppm) were selected as per the OECD guidelines.¹³

Evaluation of immune status

Humoral immune response

To study the humoral immune response, hemagglutination test (HA) and levels of serum immunoglobulins (IgA, IgG, IgM) by standard commercial kits were done in rats of all the four groups.

Hemagglutination test (HA)

Five rats from each group were sensitized with sheep red blood cells (SRBC’s) by intraperitoneal injection of 0.25 ml of SRBC’s suspended in PBS (pH-7) containing 1.25 × 10⁶ cells per animal (antigen) after 8 weeks of feeding experimental feed followed by a booster dose of the antigen after 7 days. Serum was collected at the time of sacrifice, i.e. after 1 week of a booster dose. Hemagglutination (HA) was carried out by microtitration techniques.¹⁴ Briefly, the HA test was performed in the U shaped micropersplex plate. Two-fold serial dilution of serum by properly mixing at each time was prepared in phosphate-buffered saline (PBS, pH 7.4) keeping the final volume of 0.05 ml in each well except in control well, which contained PBS alone. After that 0.05 ml of 0.5%, SRBC suspension was added to all the micropersplex plate wells. A known negative control was also included. The plate was swirled gently for mixing and uniform distribution of erythrocytes and left at room temperature (20–25°C) for 40–45 min. The HA pattern (a diffused sheet of agglutinating RBC’s covering the bottom of the wells) was read with the aid of the reading lens and the titer was recorded as the reciprocal of the highest dilution showing complete agglutination of erythrocyte and expressed as log₂/0.05 ml.

Immunoglobulins assay

The humoral immune status of the experimental rats was assessed by estimating the serum immunoglobulin (IgG, IgM, IgA) levels by Enzyme-Linked ImmunoSorbent Assay (ELISA) using rat immunoglobulins quantitation kits (Rat IgG ELISA Kit-E111-128, Rat IgM ELISA Kit-E111-100, Rat IgA ELISA Kit-E111-102) (Bethyl Laboratories, Inc. USA) as per the manufacturer’s instructions. Briefly, the plates were coated with 1 µl capture antibodies with 100 µl coating buffer and were incubated at room temperature (20–25°C) for 60 min. After incubation, plate wells were washed with the washing solution three times. After washing, the blocking solution was added to each well and incubated at room temperature for 30 min. The blocking solution was removed and plate wells were washed three times with the washing solution. The standards were diluted with a sample according to the manufacturer’s recommendation. The serum samples were diluted 50 times with sample diluent to fall within the concentration range of the standards. Then 100 µl of standards and samples were transferred to the respectively assigned wells and incubated at room temperature for 60 min. After incubation, the samples and standards were removed and the plates were washed with washing solution five times. The horseradish peroxidase (HRP) conjugates were diluted in conjugate diluent as per the manufacturer’s recommendation (1:50,000), and 100 µl of diluted HRP conjugates were transferred to each well and incubated at room temperature for 60 min. Then plates were washed five times with washing solution. Tetramethyl benzidine (TMB) substrate solution was prepared according to the manufacturer’s recommendation by mixing equal volumes of the two-substrate reagents provided in the kit and then 100 µl of substrate solution was transferred to each well and incubated for 30 min. To stop the TMB reaction, 100 µl of 2 M H₂SO₄ was added to each well and immediately the optical density (OD) values were recorded in a microtitre plate reader (BioRad, California, USA) at 450 nm wavelength. For the calculation of results, the duplicate readings were averaged for each standard (as given above) and sample. The standard curve was drawn for each set of samples using computer software. The concentration of each immunoglobulin was calculated from the standard curve.

Cell-mediated immune response

To evaluate the cell-mediated immune response, DTH response to ovalbumin, lymphocyte stimulation index, analyses of CD4⁺ and CD8⁺ T lymphocytes, and mRNA expression levels of selected cytokines like IL-2, IFN-γ, IL-4 and IL-10 by quantitative Real-time PCR were done in all treatment groups.

Delayed type hypersensitivity test (DTH)

For the DTH test, primary immunization was done 8th week after feeding experimental feed. Rats of the treatment group were injected with 10 µg ovalbumin (OVA) in 20 µl PBS intradermally in the right ear pinna. The left ear pinna was injected with 20 µl PBS alone. Secondary immunization was done with the same doses on the 15th day of primary immunization. DTH was assessed by measuring the thickness of ear pinna measured at 0, 24, 48, and 72 h by electronic vernier caliper (Forbes & Company Limited, Mumbai, India) and an increase in the thickness of pinna at different intervals was recorded. At each interval, the skin pieces from the ear pinnae were surgically removed after proper anesthesia and preserved in 10 percent buffered neutral formalin for histopathological examination.

Lymphocyte stimulation test (LST)

Lymphocyte stimulation test using MTT (3-(4, 5-dimethyl thiazol-2-yl) 2, 5-diphenyl tetrazolium bromide) was performed at 4th, 8th and 12th week of the experiment as per the procedure described by Bounous et al.¹⁵ After sacrificing the animal, spleen from different groups was collected in sterile condition in chilled PBS having 5% dextrose (PBS-D). Single-cell suspension from spleen was prepared by repeated perfusion with sterile PBS-D using an insulin syringe. The splenocytes were isolated using Histopaque. To 3 ml of Histopaque (1.077 g/ml, Sigma Aldrich, Saint Louis, Missouri, USA), 6 ml of a single-cell suspension of above-collected splenocytes were carefully over-layered. The tubes were centrifuged at 1400 rpm for 40 min at 40°C. The mononuclear cells (MNCs) were collected from liquid-histopaque interface and washed twice with sterile PBS-D at 800 rpm for 10 min and finally, the cell suspensions were resuspended in 1 ml of RPMI 1640 growth medium (Sigma Aldrich, Saint Louis, Missouri, USA) without having phenol red. The cell count and viability were determined by Trypan blue dye exclusion method. To calculate the number of cells per ml volume, the following formula was applied:

No. of cells/ml = Average number of cells× 10^{4} × dilution factor

The blastogenic response of splenocytes was assessed by the MTT colorimetric method.¹⁶ For stimulation, Concanavalin-A (Con-A) was used as a mitogen. A volume of 100 µl of cell suspension (2 × 10⁶ cells/ml) from different groups of rats was plated in three sets of triplicate onto 96-well flat-bottom tissue culture plate (Corning, USA), except the marginal wells which were devoid of any cell suspension. The second and third sets of triplicate culture wells received 100 µl of RPMI-GM containing 100 µg/ml of Con-A. The first set received 100 µl of sterile RPMI-GM without Con-A (negative control). The plate was sealed properly with adhesive tape and incubated at 37°C in a humidified chamber with 5% CO₂ tension for 96 h. After 96 h, 20 µl of MTT solution (5 mg/ml in PBS) was added to each well and further incubated at 37°C for 4 hr. The plates were then centrifuged at 1,500 rpm for 10 min and 100 µl of culture supernatant was discarded from each well. Finally, 150 µl of dimethyl-sulfoxide (DMSO) was added to each well and mixed thoroughly avoiding froth formation to dissolve formazan crystals. The intensity of color development was measured by taking OD at 550 nm. Stimulation index (SI) was calculated as per the following formula:

{SI}_{(MTT)} = \frac{Mean OD of stimulated culture}{Mean OD of unstimulated culture}

Flow cytometry (FACS) analysis of CD4⁺: CD8⁺peripheral lymphocytes

For the enumeration of CD4⁺ and CD8⁺ T-cells, flow cytometric analysis of peripheral mononuclear cells was performed on the FACS Calibur® instrument (Becton Dickinson, San Jose, California). The monoclonal antibodies (Anti-rat CD3⁺: FITC/ CD4⁺: RPE/CD8⁺: Alexa Flour, 647) used in the study were procured from AbD SeroTech (UK). The peripheral blood mononuclear cells (PBMC) for cytometry were obtained at the 4th, 8th, and 12th weeks of the experiment as per the manufacturer’s instructions. Briefly, 50 µl blood was collected in sterilized vials containing EDTA. Anti CD4⁺ and CD8⁺ rat conjugates were added in the concentration of 1µg/millions of cells (10 µl conjugate/sample), vortexed, and incubated in dark for 20–30 min. The cells were washed twice with 1 ml of PBS (pH 7.4) and spun at 4000 rpm for 5 min. After this, RBC lysis buffer (10 times the volume of blood) was added, vortexed, and kept at room temperature for 7–10 min. The lysate was centrifuged at 4000 rpm for 5 min and the supernatant was discarded. The cell pellet was washed with PBS and dissolved in 500 µl of PBS. After vortexing, the immunophenotyping was done with Fluorescence Activated Cell Sorter (FACS) Calibur, and the results were analyzed in the Cell Quest^TM program of FACS Calibur (Becton Dickinson).

Expression of cytokines by quantitative real-time PCR (qRT-PCR)

Selected cytokines (IL-2, IFN-γ, IL-4, and IL-10) expression studies in spleen samples were carried out by quantitative Real-Time PCR (MX 3000 P System Stratagene, USA). Fresh spleen tissue samples were collected from rats of all the groups, aseptically and RNA was extracted using the TRIzol reagent (Life Technologies, USA) method as per the manufacturer’s instructions. The extracted RNA was used to synthesize cDNA using Reverse Transcription System (Promega, USA). The mRNA expression levels of cytokines by qRT PCR was done by using specific primers (Table 1). All reactions were set in 20 µl reaction mix in optical 96-well reaction plates. The reaction mixture consisted of 10 µl Maxima™ SYBR Green/ROX qPCR Master Mix (2x) (Fermantas, USA) (containing SYBR Green I dye, ROX Passive reference dye, MaximaTM Hot start Tag DNA polymerase, dNTP in optimized PCR buffer and dUTP), a 1 µl of the respective each cytokine gene forward and reverse primers, a 1 µl of cDNA template (∼50 ng final concentration) and volume was made up to 20 µl with nuclease-free water. The qRT PCR reactions were run with following cycling conditions for all the mentioned cytokine genes. The reactions started with initial incubation at 94°C for 5 min followed by 40 cycles of amplification with denaturation at 94°C for 30 sec, followed by annealing at 55°C (IFN-γ, IL-4, and IL-10) and 56°C (IL-2) for 30 sec and extension at 72°C for 30 sec each. The housekeeping gene, β-actin, was run at the initial incubation of 94°C for 5 min followed by 40 cycles of amplification with denaturation at 94°C for 30 sec, followed by annealing at 56°C for 30 sec and extension at 72°C for 30 sec each, and dissociation curve was generated at temperatures 56°C and 94°C. To check the quality of PCR products, an analysis of a melting curve followed by agarose gel electrophoresis was performed after each run. The result was expressed as the threshold cycle values (Ct). The data obtained were analyzed using the 2^–(ΔΔCt) method.¹⁹

Table 1.

List of primers used for amplification of different genes by qRT PCR.

Primer name	Sequence (5′ →3′)	Product (bp)	Annealing temp. (°C)	Reference
IL-2	F-CCCCATGATGCTCACGTTTA R-ATTTTCCAGGCACTGAAGATGTTT	76	56	¹⁷
IL-4	F-ACCTTGCTGTCACCCTGTTCTGC R-GTTGTGAGCGTGGACTCATTCACG	352	55	¹⁸
IL-10	F-TGCCAACCCTTGTCAGAAATGATCAAG R-GTATCCAGAGGGTCTTCAGCTTCTCTC	127	55	¹⁸
IFN-γ	F-AGTCTGAAGAACTATTTTAACTCAAG TAGCAT R-CTGGCTCTCAAGTATTTTCGTGTTAC	117	55	¹⁷
β-actin	F-AGAAGAGCTATGAGCTGCCTGACG R-CTTCTGCATCCTGTCAGCCTACG	236	56	¹⁸

Pathomorphological studies of lymphoid organs

Gross pathology

Rats from each group were euthanized at regular intervals viz., 2, 4, 6, 8, 10, and 12 weeks and the carcasses were weighed individually to record the body weights. A systematic necropsy examination of the sacrificed rats was conducted immediately. All the lymphoid organs were thoroughly examined in situ and visible gross alterations were recorded.

Relative organ weights

The spleen and thymus were weighed individually at every sacrifice interval and their relative weights were calculated as the percent body weight.

Histopathology

After thorough gross examination, small representative pieces of spleen, thymus, and Peyer’s patches (less than 5 mm thickness) were collected in a 10% neutral buffered formalin solution. After 3–4 days of fixation, the tissues were washed in running water for 7–8 h, dehydrated in ascending grades of ethyl alcohol, cleared in benzene, and embedded with melted paraffin wax (melting point 58°C). The paraffin blocks were prepared and the sections were cut at 4–5 µm thickness with a hand-operated microtome. The paraffin-embedded sections were then passed through sequential steps of deparaffinization in xylene, rehydration by passing through descending grades of ethyl alcohol to running tap water and stained by routine hematoxylin and eosin stain.²⁰ The stained tissue sections were examined under the microscope for assessing histopathological alterations.

Statistical analyses

Data of various parameters were presented as Mean + standard error (S.E.) of the mean for each group. One-way/two-way analysis of variance (ANOVA) was used to compare differences between control and treatment groups and the means were compared with Duncan post hoc using the SPSS software (version 16.0) and a value of P < 0.05 was taken as significant.

Result

Mortality pattern

In the present study, mortality recorded in different T-2 toxin treated group rats were 6.25% in 0.5 ppm, 8.33% in 0.75 ppm, and 12.00% in 1.0 ppm group in a dose-dependent manner during the experimental period.²¹

Humoral immune response

The humoral immune response status was assessed by measuring the HA titer levels against sheep RBCs in terms of log (2)/0.05 ml and also the levels of different classes of immunoglobulins. The HA titers were significantly decreased (p < 0.05) in all treatment groups as compared to that of the control group at 70th day and were 3.75 ± 0.25, 2.25 ± 0.25, 1.50 ± 0.29 and 4.75 ± 0.25, respectively in 0.5, 0.75, 1.0 ppm T-2 fed and control group rats. Among the treatment groups, there was a dose-dependent decrease in HA titers. The mean values of different classes of immunoglobulins have been presented in Table 2. The levels of all the three classes of immunoglobulins (IgG, IgM, and IgA) in serum estimated at 4th, 8th, and 12th weeks showed a dose and duration-dependent decrease. This decrease was significant (p < 0.05) in toxin fed rats when compared with that in the control group except at 4 weeks where the levels of IgG and IgM immunoglobulins in least toxin fed and the control group rats were more or less comparable.

Table 2.

Effect of T-2 on immunoglobulin levels (µg/ml) in rats of different treatment groups.

Type of immunoglobulin	Intervals of sacrifice	Treatment groups
Type of immunoglobulin	Intervals of sacrifice	GI	GII	GIII	GIV
IgG (µg/ml)	4 wk	1010.333^bBC ± 33.122	992.000^aAB ± 42.675	966.668^aA ± 23.570	1024.000^bC ± 24.833
	8 wk	987.668^bB ± 23.932	933.333^bAB ± 23.570	883.333^aA ± 31.181	1027.333^cC ± 30.093
	12 wk	956.333^cC ± 20.001	876.000^bB ± 27.653	831.668^aA ± 22.392	1033.000^dD ± 41.294
IgM (µg/ml)	4 wk	300.233^bcBC ± 0.125	291.813^abAB ± 3.272	281.088^aA ± 2.095	310.733^cC ± 6.810
	8 wk	281.523^bB ± 6.947	263.153^aAB ± 1.812	238.040^aA ± 13.735	313.070^cC ± 9.426
	12 wk	252.058^cC ± 3.862	236.788^bB ± 1.218	206.158^aA ± 4.952	312.193^dD ± 6.689
IgA (µg/ml)	4 wk	13.811^bcBC ± 0.250	11.968^bAB ± 1.276	9.960^aA ± 0.368	15.228^Dc ± 0.272
	8 wk	12.177^cB ± 0.359	9.908^bAB ± 0.883	8.079^aA ± 0.347	15.913^dC ± 0.431
	12 wk	10.178^Cc ± 0.300	8.975^bB ± 0.128	7.727^Aa ± 0.336	14.410^dD ± 0.592

Data are presented as Mean + S.E. (n = 8 rats/group). Means bearing at least one common superscript (a, b, c, d and A, B, C, D) do not differ significantly between groups and weeks, respectively (P < 0.05). IgG/IgM/IgA: Immunoglobulin G, M, A. Group I (0.5 ppm), Group II (0.75 ppm), Group III (1.0 ppm), Group IV (Control).

Cell-mediated immune response

The cell-mediated immune response was evaluated employing DTH reaction to ovalbumin, assessment of lymphocyte stimulation indices, analysis of CD4⁺ and CD8⁺ in peripheral blood lymphocytes and expression of cytokines by Quantitative Real-Time PCR (qRT-PCR).

DTH to ovalbumin : Grossly, the ear pinna revealed hot and painful focal swelling at 24 h, which tended to decrease by 48 and 72 h in the rats of all the groups. The skin thickness measurements in various groups at various intervals are presented in Table 3. The increase in skin thickness showed significant differences at all intervals among various groups being least at 72 h in 1.0 ppm T-2 fed rats (0.390 ± 0.024 mm) when compared with lower treatment groups and control group. Histopathological examination of skin biopsies collected at 24, 48 and 72 h, revealed varying degrees of acute inflammatory reaction characterized by intense infiltration of polymorphs and mononuclear cells (MNCs) which extended deep into subchondral muscles with dermal edema and vascular congestion at 24 h in group IV (control) rats (Figure 1). The intensity of inflammation was increased at 48 h with a predominance of large mononuclear cells and fibroplasias and then became milder at 72 h. The inflammatory response in 0.5, 0.75, and 1.0 ppm T-2 fed rats followed the almost similar pattern of increase up to 48 h to that of the control group in a dose and duration-dependent manner. The inflammatory response subsided at 72 h and the infiltrating cells were barely visible in the tissue section at 72 h in 1.0 ppm T-2 fed rats. The inflammatory response was mildest in 1.0 ppm T-2 fed rats when compared with lower treatment groups and control group at all intervals (Figure 1(a) to (h)).

Table 3.

Effect of T-2 on ovalbumin-induced delayed-type hypersensitivity response evaluated by an increase in skin thickness (mm) in rats of different treatment groups at 10 weeks of treatment.

Time interval	GI	GII	GIII	GIV
24 h (mm)	0.750 ± 0.020^c	0.583 ± 0.0^b	0.417 ± 0.031^a	0.867 ± 0.024^d
48 h (mm)	0.773 ± 0.021^c	0.613 ± 0.025^b	0.437 ± 0.031^a	0.973 ± 0.008^d
72 h (mm)	0.730 ± 0.019^c	0.553 ± 0.027^b	0.390 ± 0.024^a	0.750 ± 0.020^c

Data are presented as Mean + S.E. (n = 8 rats/group). Means bearing at least one common superscript (a, b, c, d) do not differ significantly between groups (P < 0.05). h: hours. Group I (0.5 ppm), Group II (0.75 ppm), Group III (1.0 ppm), Group IV (Control).

Figure 1.

Effects of T-2 on ovalbumin-induced DTH response in skin in rats of different treatment groups at 10 weeks. (a) Group IV, 24 h: intense acute inflammatory reaction with dense polymorphs and MNCs infiltration (star), vascular congestion, and edema (arrow) (H&E ×100). (b) Group I, 24 h: intense inflammatory reaction with the predominance of polymorphs (star), edema (arrow), and vascular engorgement (arrowhead) (H&E ×100). (c) Group II, 24 h: diffuse inflammatory reaction (star), with edema (arrow) in both epi and subchondral connective tissue (H&E ×100). (d) Group III, 24 h: mild cellular infiltration (star) and edema (arrow) (H&E ×100). (e) Group IV, 72 h: sparse number of inflammatory cells with little edema (arrow) in both epi and subchondral connective tissue (H&E ×100). Higher magnification: dermis showing infiltration of macrophages (arrowhead), lymphocytes (star) and fibroblast (hollow arrow) proliferation (H&E ×400). (f) Group I, 72 h: Dermis showing less number of inflammatory cells with fibroblast proliferation (arrow) (H&E ×100). (g) Group II, 72 h: low inflammatory reaction with loosening of tissue (H&E ×100). (h) Group III, 72 h: Dermis showing practically normal appearance (H&E ×100). GI: 0.5 ppm, GII: 0.75 ppm, GIIII: 1.0 ppm, GIV: control. h: hour.

LST: The SI, following Con-A induced stimulation of lymphocytes collected from rats of all the groups at 4th, 8th, and 12th weeks of the trial are presented in Table 4. The SI was significantly lower (P < 0.05) in all the treatment groups at all intervals as compared to those in the control group at corresponding intervals of T-2 treatment. Among treatment groups, the SI was lowest in 1.0 ppm T-2 fed rats.

Table 4.

Effect of T-2 on lymphocyte stimulation index (SI) in rats of various treatment groups.

Intervals of sacrifice	GI	GII	GIII	GIV
4 wk	0.821 ± 0.083^bB	0.774 ± 0.032^abC	0.630 ± 0.030^aC	1.049 ± 0.053^c
8 wk	0.721 ± 0.001^cB	0.546 ± 0.015^bB	0.460 ± 0.007^aB	0.984 ± 0.012^d
12 wk	0.698 ± 0.003^cA	0.452 ± 0.069^bA	0.310 ± 0.004^aA	1.044 ± 0.017^d

Data are presented as Mean + S.E. (n = 8 rats/group). Means bearing at least one common superscript (a, b, c, d and A, B, C, D) do not differ significantly between groups and weeks, respectively (P < 0.05). wk: week. Group I (0.5 ppm), Group II (0.75 ppm), Group III (1.0 ppm), Group IV (Control).

Analysis of CD4⁺ and CD8⁺peripheral lymphocytes by FACS : The percentage of CD4⁺ and CD8⁺ lymphocytes in all treatment groups were significantly lower (P < 0.05) than control (Table 5). Similarly, CD4⁺: CD8⁺ ratio showed significant (P < 0.05) variation among groups. At 4th and 8th weeks, CD4⁺: CD8⁺ cell ratios in 0.75 and 1.0 ppm T-2 fed rats were comparable to each other but were significantly lower than those of 0.5 ppm T-2 fed and control group rats. At 12th week, all treatment groups showed significantly lower CD4⁺: CD8⁺ cell ratio being lowest in 1.0 ppmT-2 fed rats (1.271 ± 0.018) followed by 0.75 and 0.5 ppm T-2 fed rats (1.492 ± 0.204 and 1.905 ± 0.147, respectively) as compared to that in the control group (2.965 ± 0.065).

Table 5.

Effect of T-2 on the percentage of peripheral lymphocyte subpopulation and CD4⁺: CD8⁺ ratios in rats of various treatment groups.

Intervals of sacrifice	Cell surface receptor	GI	GII	GIII	GIV
4 wk	CD4⁺ (%)	45.043^bB ± 3.5661	42.233^bC ± 1.0463	31.167^aC ± 1.4721	55.390^cC ± 1.7082
	CD8⁺ (%)	25.333^bB ± 2.667	19.497^aB ± 2.302	16.047^aB ± 1.264	28.783^cC ± 3.125
	CD4⁺: CD8⁺	2.892^bB ± 0.076	2.080^aA ± 0.028	1.817^aA ± 0.127	2.972^cC ± 0.616
8 wk	CD4⁺ (%)	41.763^cB ± 0.590	33.270^bB ± 1.143	21.883^aB ± 1.790	51.083^d ± 2.835
	CD8⁺ (%)	20.207^bA ± 0.408	13.468^aA ± 1.932	9.360^aA ± 0.349	27.350^c ± 1.038
	CD4⁺: CD8⁺	2.338^b ± 0.103	1.966^a ± 0.071	1.554^a ± 0.046	2.942^c ± 0.286
12 wk	CD4⁺ (%)	37.610^cA ± 4.495	25.217^bA ± 1.116	15.590^aA ± 0.382	52.433^d ± 1.730
	CD8⁺ (%)	19.560^cA ± 0.885	13.197^bA ± 0.926	8.028^aA ± 2.041	27.893^d ± 0.850
	CD4⁺: CD8⁺	1.905^c ± 0.147	1.492^b ± 0.204	1.271^a ± 0.018	2.965^d ± 0.065

Data are presented as Mean + S.E. (n = 8 rats/group). Means bearing at least one common superscript (a, b, c, d and A, B, C) do not differ significantly between groups and weeks, respectively (P < 0.05). Group I (0.5 ppm), Group II (0.75 ppm), Group III (1.0 ppm), Group IV (Control).

Expression of cytokines by qRT-PCR : After each complete reaction in real-time PCR, the data were stored along with amplification plots and dissociation curves for each cytokine. The relative quantity of each cytokine was calculated after normalizing with the β-actin housekeeping gene (Table 6).

Table 6.

Relative quantities of mRNA expression of different cytokines in the spleen of rats.

mRNA expression of cytokine	Intervals of sacrifice	GI	GII	GIII	GIV
IL-2	4 wk	0.967^b ± 0.003	0.957^a ± 0.003	0.953^a ± 0.003	1.000^c ± 0.000
	8 wk	0.957^b ± 0.003	0.943^a ± 0.003	0.943^a ± 0.003	1.000^c ± 0.000
	12 wk	0.933^b ± 0.003	0.930^b ± 0.006	0.913^a ± 0.003	1.000^c ± 0.000
INF-γ	4 wk	0.953^b ± 0.003	0.937^a ± 0.003	0.930^a ± 0.006	1.000^c ± 0.000
	8 wk	0.940^b ± 0.000	0.927^a ± 0.003	0.920^a ± 0.006	1.000^c ± 0.000
	12 wk	0.923^c ± 0.009	0.900^b ± 0.006	0.867^a ± 0.009	1.000^d ± 0.000
IL-4	4 wk	0.957^b ± 0.003	0.943^a ± 0.003	0.940^a ± 0.000	1.000^c ± 0.000
	8 wk	0.943^c ± 0.003	0.930^b ± 0.006	0.913^a ± 0.003	1.000^d ± 0.000
	12 wk	0.937^c ± 0.003	0.910^b ± 0.006	0.880^a ± 0.012	1.000^d ± 0.000
IL-10	4 wk	0.997^b ± 0.003	0.987^a ± 0.003	0.987^a ± 0.003	1.000^b ± 0.000
	8 wk	1.033^a ± 0.033	1.167^b ± 0.033	1.300^c ± 0.058	1.000^a ± 0.000
	12 wk	1.167^ab ± 0.088	1.267^bc ± 0.033	1.467^c ± 0.088	1.000^a ± 0.000

Data are presented as Mean + S.E. (n = 8 rats/group). Means bearing at least one common superscript (a, b, c) do not differ significantly between groups (P < 0.05). IFN-γ: Interferon gamma; IL-2/IL-4/IL-10: Interleukin 2,4,10. Group I (0.5 ppm), Group II (0.75 ppm), Group III (1.0 ppm), Group IV (Control).

IL-2: The levels of IL-2 mRNA expression were significantly lowered in T-2 intoxicated groups at 4th, 8th, and 12th weeks as compared to its level in the control animals at the corresponding intervals. Rats fed T-2 toxin at 0.75 and 1.0 ppm showed comparable levels, but significantly lower than that of 0.5 ppm T-2 fed and control group rats at 4 and 8 weeks of the experiment. At the 12th week, the lowest IL-2 mRNA expression was recorded in 1.0 ppm T-2 fed rats, followed by 0.75 and 0.5 ppm T-2 fed rats as compared to the control group.

IFN-γ: The IFN-γ mRNA expression was significantly lower (P < 0.05) in all treatment groups than the control group in a dose and a duration-dependent manner. At 4 and 8 weeks, 0.75 and 1.0 ppm T-2 fed rats showed comparable levels among themselves, but significantly lower than that in 0.5 ppm T-2 fed and control group rats.

IL-4: The levels of IL-4 mRNA expression were significantly lower in the treatment groups as compared to those in the control group in a dose and time of duration of the T-2 feeding dependent manner. All treatment group rats showed a significant difference in IL-4 expression among themselves at the 8th and 12th weeks.

IL-10: The level of IL-10 mRNA expression among the lowest toxin fed group rats as well as control groups did not reveal any significant (P < 0.05) variation at different time intervals, but 0.75 and 1.0 ppm T-2 fed rats showed significantly higher levels than 0.5 ppm T-2 fed and control group rats at all intervals. At the end of the trial, the highest expression of IL-10 was recorded in 1.0 ppm T-2 fed rats.

Pathomorphological studies of lymphoid organs

Gross pathology

In comparison to control group rats, small-sized hypoplastic spleens and thymuses were seen in all treatment group animals in a dose-dependent manner from the 8th week onward. At the 12th week, a severe reduction in the size of the spleen and thymus was observed in treatment groups in a dose-dependent manner (Figure 2).

Figure 2.

Effects of T-2 toxin on lymphoid organs (gross) in rats of different treatment groups at 12 weeks. Dose-dependent decrease in size of (a) spleen and (b) thymus at 12 weeks of T-2 treatment (GI: 0.5 ppm, GII: 0.75 ppm, GIIII: 1.0 ppm, GIV: control).

Relative organ weights

Spleen

The relative spleen weights of the toxin fed groups were lower than those of the control at all intervals, but the decrease was not significant statistically at the 4th week (Table 7). After the 8th week, all treatment groups showed a significant dose-dependent decrease in relative spleen weights when compared with that in the control group at 10th and 12th weeks post-feeding of T-2 toxin.

Table 7.

Effect of T-2 on relative weights of different organs (% of bw) in rats of various treatment groups.

Lymphoid organ	Treatment groups	2 wk	4 wk	6 wk	8 wk	10 wk	12 wk
Spleen (% of bw)	GI	0.337^B ± 0.054	0.331^abB ± 0.026	0.312^bAB ± 0.061	0.298^bA ± 0.128	0.281^cA ± 0.131	0.233^cA ± 0.076
	GII	0.330^C ± 0.056	0.307^abBC ± 0.061	0.293^abAB ± 0.034	0.251^aA ± 0.040	0.223^bA ± 0.097	0.183^bA ± 0.044
	GIII	0.322^E ± 0.060	0.288^aD ± 0.062	0.251^aC ± 0.063	0.203^aB ± 0.052	0.149^aAB ± 0.032	0.107^aA ± 0.004
	GIV	0.340^A ± 0.051	0.354^aA ± 0.041	0.398^cA ± 0.046	0.441^cA ± 0.058	0.469^dAB ± 0.073	0.502^dB ± 0.087
Thymus (% of bw)	GI	0.210^D ± 0.032	0.180^abC ± 0.016	0.169^bC ± 0.047	0.155^bB ± 0.013	0.138^cA ± 0.043	0.115^cA ± 0.005
	GII	0.202^E ± 0.033	0.167^abE ± 0.028	0.147^aD ± 0.021	0.131^aC ± 0.012	0.108^bB ± 0.033	0.089^bA ± 0.020
	GIII	0.192^F ± 0.030	0.144^aCE ± 0.020	0.112^aD ± 0.007	0.090^aC ± 0.016	0.070^aB ± 0.031	0.053^aA ± 0.034
	GIV	0.220^a ± 0.047	0.222^b ± 0.038	0.231^c ± 0.047	0.246^c ± 0.063	0.255^d ± 0.042	0.260^d ± 0.058

Data are presented as Mean + S.E. (n = 8 rats/group). Means bearing at least one common superscript (a, b, c, d and A, B, C, D, E) do not differ significantly between groups and weeks, respectively (P < 0.05). bw: body weight. Group I: 0.5 ppm, Group II: 0.75 ppm, Group III: 1.0 ppm, Group IV: Control.

Thymus

The relative thymus weights in the toxin fed groups were lower than those in the control at all intervals in a dose-dependent manner (Table 7). The highest toxin fed group showed significantly reduced thymus weights as early as the 4th week in comparison with the control group. At 6th and 8th weeks, 0.75 and 1.0 ppm T-2 fed rats showed comparable weights, but significantly reduced in comparison with 0.5 ppm T-2 fed and control group rats. After the 8th week, all treatment groups showed significantly decreased relative weights. Lowest thymus weights were recorded in 1.0 ppm T-2 fed rats, followed by 0.75 and 0.5 ppm T-2 fed rats as against those in the control group at 10th and 12th weeks post-feeding of T-2 toxin.

Histopathology

Spleen

In 0.5 ppm T-2 fed rats, spleens remained normal for up to 4 weeks. At 6 weeks, multinucleated megakaryocytes with thinly populated splenic follicles (Figure 3(a)) were observed. Similar types of changes with increasing magnitude were seen in the spleen at successive intervals. At 12 weeks, lymphocytes in the follicles were depleted, as evidenced by the presence of irregular empty spaces with condensed nuclear fragments giving a starry sky appearance to lymphoid follicles (Figure 3(b)). The spleen in 0.75 ppm T-2 fed rats also remained normal for up to 2 weeks. At 4 and 6 weeks, engorgement of the splenic blood vessel with mild red pulp congestion (Figure 3(c)) and mild depletion of lymphocytes giving the starry sky appearance with dark condensed nuclear bodies was observed in three animals. Similar types of changes with increasing severity were observed at 8–12 weeks of T-2 feeding. Rats fed T-2 toxin at 1.0 ppm revealed the normal appearance of spleen at an early interval (2 weeks). At 4 weeks, thinly populated lymphocytes in splenic follicles with the presence of a large number of multinucleated megakaryocytes were observed. At the 6th week, depletion and lymphocytolysis around the splenic arterioles and in lymphoid follicles were observed. Similar types of changes with an increase in severity were observed at 8–12 weeks of T-2 feeding. Scattered foci of lymphocytic depletion with the presence of condensed nuclear bodies (Figure 3(d)) and severe lymphocytolysis (Figure 3(e)) were seen in the majority of animals. Control group animals revealed well developed splenic corpuscles with densely packed lymphocytes (Figure 3(f)).

Figure 3.

Effects of T-2 toxin on the histopathological architecture of spleen in rats of different treatment groups at various intervals. Group I: (a) Thinly populated lymphocytes in splenic follicle and presence of multinucleated megakaryocytes (arrow) at 6th wk (H&E ×400) and (b) irregular empty spaces with presence of condensed nuclear bodies (arrow) at 12th wk (H&E ×400). Group II: (c) engorgement of splenic blood vessel with mild red pulp congestion at 6th wk (H&E ×100). Group III: (d) multiple variable-sized empty spaces having condensed nuclear fragments (arrow) at 10th wk (H&E ×400) and (e) severe depletion and cytolysis of lymphocytes around the splenic blood vessels at 12th wk (H&E ×400). Group IV: (f) well developed spleenic follicle with a good lymphoid population (H&E ×400). GI: 0.5 ppm, GII: 0.75 ppm, GIIII: 1.0 ppm, GIV: control. wk: week.

Thymus

The changes in the thymus were almost similar to those observed in the spleen but were of more severity in terms of single-cell apoptosis in the form of tiny empty spaces containing condensed nuclear fragments that coalesced to form large foci of lymphocytolysis. The 0.5 ppm T-2 fed rats showed normal architecture up to 4 weeks (Figure 4(a)) while 0.75 ppm T-2 fed rats showed thymic changes earlier than seen in 0.5 ppm T-2 fed rats and characterized by few spaces of lymphocytic depletion (Figure 4(b)) at 2 weeks. Similarly, 1.0 ppm T-2 fed rats revealed more severe lesions at an early interval (2 weeks) as compared to 0.5 and 0.75 ppm T-2 fed rats and characterized by moderate to severe lymphocytic depletion in the thymic follicle (Figure 4(c)). In 0.5 ppm T-2 fed rats after 4 weeks onward, mild to moderate depletion of lymphocytes in the thymic follicles were observed. Similar changes, but of more severity were observed at successive periods of trial. Similar types of lesions in 0.75 ppm T-2 fed rats with increased severity were noticed with the progression of time, being the most severe at 8th and 12th weeks of T-2 feeding. T-2 toxin fed rats at 1.0 ppm level apart from these lesions in more severe nature than observed in 0.5 and 0.75 ppm T-2 fed rats also showed intrafollicular hemorrhage at 10 weeks (Figure 4(d)). At 12 weeks, more chronic types of changes were seen as evidenced by the extensive proliferation of interfollicular connective tissue with atrophy of thymic lobules (Figure 4(e)) which became irregular and constricted. Group IV (control) animals did not reveal appreciable histopathological lesions in thymus throughout the period of the experiment.

Figure 4.

Effects of T-2 toxin on the histopathological architecture of thymus and Peyer’s patches in rats of different treatment groups at various intervals. (a) Thymus: good lymphocytes population in cortex and medulla in group I at 4th wk (H&E ×400) while earlier thymic changes than seen in group I and characterized by few spaces of lymphocytic depletion (stars) in group II at 2nd wk (b) (H&E ×400). Group III animals (d) revealed more severe lesions at an early interval (2nd wk) as compared to group I and II and characterized by moderate to severe lymphocytic depletion in thymic follicle with fragments of condensed nuclear material (star) (H&E ×400), (d) intrafollicular hemorrhage (arrow) at 10th wk (H&E ×100) and (e) extensive proliferation of interfollicular connective tissue (arrow) with atrophy of thymic lobule at 12th wk (H&E ×40). (f) Peyer’s patches: multiple variable-sized empty spaces (stary sky appearance) having condensed nuclear fragments (arrow) in group II at 12th wk (H&E x400) and (g) more or less similar lesions (arrow) of more severity with the progression of time were observed in group III at 8th wk (H&E ×400). Group IV: (h) Well developed Peyer’s patches densely populated with lymphocytes (H&E ×400). GI: 0.5 ppm, GII: 0.75 ppm, GIIII: 1.0 ppm, GIV: control. wk: week.

Peyer’s patches

The Peyer’s patches showed changes in high T-2 toxin fed 0.5 and 1.0 ppm rats from the 4th week onward. In 0.75 ppm fed T-2 rats, prominent lesions were observed at 12 weeks, which included multiple tiny spaces depleted off lymphocytes except for the presence of nuclear remnants (apoptotic bodies) giving a starry sky appearance to lymphoid follicles (Figure 4(f)). More or less similar lesions of more severity with the progression of time were observed in 1.0 ppm T-2 fed rats at the 8th wk (Figure 4(g)). Depletion and the presence of nuclear remnants in empty spaces were observed at 10 and 12 weeks in 0.75 ppm T-2 fed rats while the 0.5 ppm T-2 fed rats animals showed comparable histologic details with that seen in the control group (Figure 4(h)) at all intervals except at 12th week.

Discussion

The experimental animals (Wistar rats) were given T-2 toxin in diet realistically and naturally of receiving the contaminated feed and fodders. OECD also recommended that the test substance should be administered orally (in diet, drinking water, or gavage) unless other routes of administration (e.g. dermal or inhalation) were considered necessary for certain specific purposes. Three doses of T-2 toxin (0.5, 0.75, and 1.0 ppm) in the present study were selected as per the guidelines of OECD which recommended that at least three dose levels and a concurrent control group should be used for toxicopathological studies. The highest dose level chosen should not be lethal or cause severe suffering to the recipient. Descending dose levels were necessary to demonstrate dosage related effect, if any. The dose selection criteria for the T-2 in the present study were factually based on the 1/10th to1/20th of the oral lethal (LD50 i.e. 2.8–3.8 mg kg⁻¹ body weight)²² dose of the T-2 in rats keeping in view the body weight and daily feed consumption.

Mortality pattern

The mortality recorded were 6.25% (0.5 ppm), 8.33% (0.75 ppm), and 12.00% (1.0 ppm) in T-2 treated animals and was attributed to fatal kidney damage and hepatotoxicity as was evidenced histologically.²³ McKean et al. reported 100% mortality inT-2 treated rats fed at a level of 4.64 and 10 mg/kg body weight (bw) within 24 hours. However, no death was recorded at a dose of 2.15 mg/kg bw.²⁴

Humoral immune response

The mean of HA titers of treatment group rats showed significantly lower levels than those observed in the control group. Rosenstein et al. found a significant decrease in thymus weight and anti-SRBC antibody titers in a dose-dependent manner in Swiss IC mice by intraperitoneal administration of T-2 toxin at 0.75 mg/kg bw per day for 7 days.²⁵ Similar decreases in antibody response to SRBC’s in mice exposed to T-2 was reported.²⁶ The ability of T-2 toxin to inhibit the protein synthesis might either be due to the destruction of polysomes or inhibition of peptidyl transferase.²⁷ In the present investigation, a significant decline in antibody titers against SRBC’s in the T-2 fed group rats might be due to a significant depletion of lymphocytes from lymphoid organs as observed histopathologically in spleen, thymus and Peyer’s patches. The HA response to SRBC’s, thymus-dependent antigens, required T and B cell co-operation for antibody synthesis.²⁸ Alternatively, the decrease in HA titers might be attributed to the decreased synthesis of immunoglobulins²⁹ as observed by a significantly reduced level of all-important classes of serum immunoglobulin (IgG, IgM, and IgA) in toxin fed groups as compared to control group animals in the present work. Tomar et al. reported that the antibody-producing ability of mouse spleen cells was significantly reduced following subacute dietary exposure to T-2 toxin.³⁰ Islam et al. found that T-2 toxin caused a reduction in circulating B and T lymphocytes as well as circulating IgG and IgM levels in female BALB/c mice after a single intraperitoneal dose of 1.8 and 3.5 mg T-2 /kg bw.³¹ Decrease in antibody synthesis in T-2 treated animals might also be due to a significant depletion of B-cell precursors i.e. CD44^low and CD45^low in bone-marrow cells.²⁸ IFN-γ controls immunoglobulin isotype switching in B lymphocytes and inhibits the proliferation of Th2 cells by shifting the immune response toward Th1 cells,³² therefore a decrease in IFN-γ mRNA expression observed in the present study also pointed to disruptions in immunoglobulin class switching.

Cell-mediated immune response

A significantly lowered CMI response was obtained by lower peripheral lymphocyte stimulation (SI) in a dose and duration manner of T-2 toxin feeding. The findings of the present work are also in line with previous reports where the decrease in SI was reported in mice.²⁶ Friend et al. reported significantly lower proliferative response of the spleen lymphocytes to Con-A and LPS in male Swiss mice fed T-2 toxin @ 20 mg/kg for 7 days³³ and in lambs treated with 0.6 mg/kg bw of T-2 toxin daily for 7 days.³⁴ Similarly, a decrease in SI by using different mitogens like Con-A, LPS, PHA, and PWM in 1.6 mg/kg bw T-2 treated mice was reported.³⁵ The amount of T-2 toxin required to inhibit lymphocyte blastogenesis (proliferation) was found to be at least 10 times less than the amount needed for inhibition of protein synthesis.³⁶ This implies that lymphocyte proliferation could be the first parameter to be affected by T-2 toxin followed by the inhibition of protein synthesis and cell death at a later stage. The ovalbumin-induced DTH also revealed results similar to SI whereby the clinical manifestations (erythema, reddening, and induration), skin thickness, and histopathological tissue reaction indicated lower immune reaction in T-2 toxin fed rats than in control. These findings are also in line with previous reports in poultry.³⁷ The mean length of survival of skin grafts from C57Bl/6 mice onto Swiss mice was 8.7 days in control recipients and 12 days in recipients treated with T-2 toxin at 0.75 mg/kg bw per day for 7 days before skin grafting and then three times a week for 20 days.²⁵ This indicated that T-2 toxin suppressed cell-mediated immunity, which resulted in the acceptance of allografts for a longer period. It was concluded that the lower DTH responses in the toxin fed animals might be due to the inhibition of the proliferation of CD8⁺ lymphocytes, the production of cytokines, which were considered to be involved in mediating delayed inflammation, and production and migration of inflammatory cells to the injection site by T-2 toxicity.

The rats in the toxin fed group showed a significant reduction in the number of CD4⁺ and CD8⁺ lymphocytes in peripheral blood. Islam et al. observed that CD4⁺ CD8⁺ double-positive subsets were decreased significantly at 24, 48, and 72 h in thymuses of mice treated with 1.75 mg/kg T-2 toxin.³⁸ Nagata et al. found that oral T-2 toxin administration at a level of 10 mg/kg to mice caused significant changes in lymphocyte subpopulation and CD4⁺ and CD8⁺ T cells were found to be the most sensitive.³⁹ Kamalavenkatesh et al. also reported a significant reduction in the number of both CD4⁺ and CD8⁺ lymphocytes in the thymus and spleen in broilers fed T-2 toxin at a level of 1 ppm for 28 days.⁴⁰ Similar results were obtained in the spleen and thymus⁴¹ and peripheral blood in broiler chickens.³⁷ On days 14 and 28, the percentages of CD4⁺ and CD8⁺ T lymphocytes in porcine ileal Peyer’s patches were lower in the experimental animals (200 µg T-2 toxin kg⁻¹ feed) than in the control group.⁴² Thus, dietary T-2 toxin was found to have a negative impact on CD4⁺ and CD8⁺ lymphocyte subpopulation as well as CD4⁺: CD8⁺ ratio and could be attributed to lymphocytopenia as previously reported by the author in T-2 toxicated animals,²¹ depletion of lymphoid elements in the immunocompetent organs and inhibition of protein synthesis by T-2 toxicity.

In the present study, IFN-γ mRNA expression was significantly reduced in toxin groups. The down-regulation of IFN-γ might be due to the inhibitory effect of dietary T-2 toxin on the blastogenesis of Th1 cells and the production of IFN-γ. Li et al. reported T-2 suppressed IFN-γ responses in Peyer’s patches to reovirus at 3 and 7 days as compared to infected controls in mice exposed intraperitoneally first to 1.75 mg T-2/kg and then 2 h later with 3 × 10⁷ plaque-forming units of reovirus serotype 1.⁴³ Ahmadi and Riazipour postulated that T-2 toxin at concentrations between 1 ng/ml and 100 ng/ml reduced the release of both IL-2 and IFN-γ in mice peritoneal macrophages and lymph node T-cells.^44,45 Obremski et al. observed a gradual decrease in the amount of IL-4 and IFN-γ cytokine transcripts throughout the experiment in porcine ileal Peyer’s patches.⁴² T-2 toxin fed animals showed down-regulated levels of IL-2 mRNA expression at all intervals. Similar findings were reported earlier in mice^43,44,45 and poultry.³⁷ Thus, lower levels of IFN-γ and IL-2 expression in T-2 treated animals suggested cellular immune suppression which was further correlated with a decreased number of CD4⁺ subset population, the source of Th1 cells secreting IFN-γ and IL-2. Similar down-regulation of IL-4 mRNA expression was noticed in the treatment group. This finding corroborated with that of the previous report.^37,43,44,45 The level of IL-10 mRNA expression among lowest toxin fed group I as well as control groups did not reveal any significant variation at different time intervals but groups II and III had higher values than both groups. This finding got support from that of Li et al.⁴³ In contrast to this finding, Ahmadi and Riazipour^44,45 and Obremski et al.⁴² reported decreased levels of IL-10 in a concentration-dependent manner (all with p < 0.01). Higher values of IL-10 promoted proliferation and terminal differentiation of immunoglobulin-secreting cells as well as down-regulated Th1 responses.⁴⁶

Reduction in relative weights and size of the spleen and thymus was seen in all treatment groups, particularly at a later stage (from 8 to 12 weeks) which was supported by histopathological findings of severe lymphocyte depletion and apoptosis, thereby lowering the cell mass and volume (atrophy) grossly and lowered weights of these organs in the present study. Similar observations were made by earlier workers in rats⁴⁷ and mice.^33,48 The changes in lymphoid organs (spleen, thymus, and Peyer’s patches) were dose and duration dependent, but of almost similar nature except that the changes were more severe in thymus than other lymphoid organs and in spleen megakaryocytosis occurred. The depletion of lymphocytes was started as single-cell apoptosis in the form of tiny empty spaces containing condensed nuclear fragments that coalesced to form large foci of lymphocytolysis, particularly in the thymus. Thymic changes also included inter and intrafollicular hemorrhage and increased interfollicular connective tissue, leading to early atrophy of thymic follicles. Similar findings had been reported earlier in mice.⁴⁸ Ueno et al. stated that T-2 toxin has radiomimetic effects specifically on actively dividing cells of the thymus, bone marrow, spleen, and lymph nodes.⁴⁹ It is possible that the proliferation of lysosomes and the increase of hydrolytic enzymatic activities such as DNAases in the thymus and spleen of T-2 treated animals could be responsible for the particular sensitivity of the lymphoid cells to T-2 toxin.⁵⁰ Kamalavenkatesh et al. reported the ultrastructural appearance of apoptotic thymocytes of T-2 treated birds characterized by shrunken and condensed nuclei, crescent margination of the chromatin against the nuclear envelop without any inflammation in T-2 toxicosis and proved that the cell death phenomenon caused by T-2 toxicosis in lymphoid organs was due to apoptosis.⁴⁰ Though specific parameters of apoptosis were not studied in the present study, the nature of the lesions developing as single cells, nuclear condensation, and fragmentation without inflammatory changes are suggestive of apoptosis in lymphoid organs. The apoptotic changes observed in lymphoid organs have also been narrated earlier and expressed possible involvement of Sphingol, p21 gene, protein kinase, cAMP signal system, and intracellular Ca²⁺ levels in apoptosis induced by T-2 toxin.⁵¹ Peyer’s patches are the largest organized lymphoid tissue in the mucosal epithelium having a significant role in mucosal immunity by activating the lymphocytes for the production of IgA antibodies. The lymphoid depletion due to apoptosis and lymphocytolysis in Peyer’s patches correlated well with reduced levels of IgA in the present study. The depletion of lymphoid cells in spleen, thymus, and Peyer’s patches suggested direct immunotoxic effects of T-2 causing reduced humoral and cellular immune responses in T-2 treated rats.

Conclusion

T-2 toxicity caused suppression of both humoral and cellular immune responses in the dose and duration-dependent manner as evidenced by decreased serum IgG, IgM, IgA levels, HA titers to SRBCs, DTH response to ovalbumin, Con-A stimulated SI, CD4⁺: CD8⁺ ratios and number of CD4⁺ and CD8⁺ lymphocytes in peripheral blood and mRNA expression levels of selected cytokines like IL-2, IFN-γ, IL-4 and IL-10 in toxicated animals. Among lymphoid organs, the thymus was found to be affected consistently more than spleen and Peyer’s patches. Considering the above findings, it was attributed that T-2 toxin has multi-immunosuppressive effects and its contamination in feeds might have long implications in field conditions in terms of vaccination failure and increased susceptibility to secondary bacterial, viral, fungal, and protozoan infections.

Footnotes

Acknowledgement

The authors are thankful to the Director, Indian Veterinary Research Institute (IVRI), Izatnagar, Bareilly, Uttar Pradesh, India, for providing financial help and necessary research facilities to carry out the present research work.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no special financial support for the research, authorship, and/or publication of this article.

ORCID iD

Shafiqur Rahman

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Immunopathological effects of experimental T-2 mycotoxicosis in Wistar rats

Abstract

Keywords

Introduction

Materials and methods

Production of T-2 toxin

Quantification of T-2 toxin

Preparation of experimental feed and diet

Experimental animals

Experimental design

Evaluation of immune status

Humoral immune response

Hemagglutination test (HA)

Immunoglobulins assay

Cell-mediated immune response

Delayed type hypersensitivity test (DTH)

Lymphocyte stimulation test (LST)

Flow cytometry (FACS) analysis of CD4+: CD8+peripheral lymphocytes

Expression of cytokines by quantitative real-time PCR (qRT-PCR)

Pathomorphological studies of lymphoid organs

Gross pathology

Relative organ weights

Histopathology

Statistical analyses

Result

Mortality pattern

Humoral immune response

Cell-mediated immune response

Pathomorphological studies of lymphoid organs

Gross pathology

Relative organ weights

Spleen

Thymus

Histopathology

Spleen

Thymus

Peyer’s patches

Discussion

Mortality pattern

Humoral immune response

Cell-mediated immune response

Conclusion

Footnotes

Acknowledgement

Declaration of conflicting interests

Funding

ORCID iD

References

Flow cytometry (FACS) analysis of CD4⁺: CD8⁺peripheral lymphocytes