Mycotoxins are the secondary metabolites of low molecular weight produced mainly by the mycelial structures of filamentous fungi, the molds, which grow as pathogens on plants or as saprophytes on stored plants and cereals¹. Acute and long term chronic toxicity (mycotoxicosis) may result in teratogenic, carcinogenic, mutagenic, and immunosuppressive effects in animals and birds on consumption of a mycotoxin contaminated diet². Direct consequences of consumption of mycotoxin(s) contaminated animal feed include: reduced feed intake, feed refusal, poor feed conversion, diminished gain in body weight, decreased disease resistance (due to immune suppression) and reduced reproductive capacities³ which lead to major economic losses.

T-2 toxin is a member of trichothecene group of mycotoxins produced by Fusarium species and is widely prevalent in environment contaminating livestock and poultry feed. Toxicodynamic studies demonstrated that T-2 toxin inhibits DNA, RNA and protein synthesis in eukaryotic cells, affects the cell cycle, and induces apoptosis both in vivo and in vitro⁴. 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 effects. Cytotoxic radiomimetic effects of T-2 toxin were considered primarily as a result of impaired protein synthesis, consequently of the inhibition of DNA and RNA synthesis⁵. The present study was therefore, conducted to elucidate clinico-haematological changes in Wistar rats given T-2 toxin in different doses at various intervals.

The rats of toxin fed groups showed variable degree of clinical signs, which included dullness, weakness, lethargy, retardation in growth, rough hair coat, disinclination to move and reduced feed intake with the intensity of clinical signs progressed with time in groups II and III. Animals in the group III (1.0 ppm T-2 toxin) started showing clinical signs from 6^th week onward. However, animals of lower dose groups (Gr. II, 0.75 ppm and Gr. I, 0.5 ppm T-2 toxin) started exhibiting clinical signs from 7^th and 8^th week onward respectively. Animals in group III (15/24) (1.0 ppm T-2 toxin) showed gangrenous dermatitis of tail at 8^th week (Fig. 1) and facial and podal dermatitis (Fig. 2) after 10^th week of T-2 toxin feeding. However, group I (0.5 ppm T-2 toxin) rats showed relatively less severe signs as compared to rest of the treated groups (Groups II and III). Control group (IV) rats remained active and alert with normal shining coat, growth rate and feed intake.

The mean body weights at 1week of toxin feeding were comparable among all the groups (Table 1). From 15 days onwards, the mean body weight of rats in the toxin fed groups exhibited slower weight gains, which were dose and duration dependent. The average weights in groups I, II, III and IV at 12^th week were 219.00±11.76, 183.85±12.23, 159.95±12.43 and 264.60±9.31 g, respectively. In control group, the body weights showed progressive and faster gains with duration as compared to those in T-2 toxin fed groups. During the period of experiment, the mortality ranged from 6.25% (group I), 8.33% (group II) and 12.00% (group III) in T-2 treated animals.

The haemoglobin concentration did not differ significantly (P<0.05) among groups at 2^nd week of T-2 toxin feeding (Table 2). At 4^th and 6^thweeks, the Hb concentration was significantly reduced in all treatment groups as compared to control group. At 10^th week post feeding, the Hb values in group I and II were comparable but significantly differed from those of group III and IV. The mean Hb values differed significantly amongst treatment groups at 12^th week post feeding being lowest in group III followed by group II and group I as compared to group IV (16.08±0.22 g/dl). The PCV values did not show any significant differences among the groups at 2^nd and 4^th weeks. However, at 6^th week, group I, II and III revealed significantly decreased PCV values as compared with that in control group and the trend continued till 12^th week post feeding. At 12^th week, lowest values were recorded in group III followed by group II and group I as compared to control group (IV). TEC values did not vary significantly among different groups at 2^nd week. At 4^th week, all treatment groups showed lower TEC values in comparison to that of control. However, at 8^th week TEC values of group I and II were comparable to each other but significantly higher than that of group III. At 12^th week, group III revealed significantly lowest (4.65±0.21×10⁶/il) TEC values followed by group II and group I as compared to that in the control group.

MCV values in T-2 toxicated groups reduced significantly in comparison to control group from 4^th week onwards. However, at 8^th week, MCV values of group I, group II were comparable but differed significantly from those of group III and group IV in a dose and duration dependent manner. At 12^th week, group III revealed significantly lowest MCV values followed by group II and group I as compared to that of the control group. MCH values at 2^nd week showed significant decrease in group III (1.0 ppm) as compared to group IV (control). At 4^th and 6^th weeks, MCH values in all treatment groups were significantly lower than that of control. At 8^th and 10^th weeks, the MCH values in group II and group III MCH values were comparable to each other but were significantly lower than those of group I and group IV. At 12^th week, all treatment groups showed significantly lower values than that of control. MCHC values in all treatment groups did not differ significantly amongst themselves upto 12^th week of T-2 toxin feeding. However, values in treatment groups were significantly lower than the control group.

Significantly (P<0.05) lower TLC values were recorded in all treatment groups as compared with the control group at all the observation intervals except 2^nd week. At 4^th week, the TLC values in group I and group IV were comparable to each other but varied significantly when compared to the values of group II and group III. However, mean TLC in group II and group III did not differ significantly upto 12^th week but significantly lower than group I and group IV. Total leucocyte counts were significantly reduced with increasing dose of T-2 and with advancing duration in the animals of groups I, II and III. The lymphocyte per cent was found to decrease significantly (P<0.05) in all treatment groups as compared with the control group at all the observation intervals except 2^nd week. At 4^th and 6^th weeks, per cent lymphocytes in group II and group III did not differ significantly but varied significantly when compared to group I and group IV, respectively. After 8^th week of T-2 toxin feeding the per cent lymphocytes differed significantly amongst the groups as well as with that of control in dose and duration manner. Lowest per cent was observed in group III followed by group II and group I at 12^th week of T-2 toxin feeding. The neutrophil per cent of group I and IV were comparable to each other but their counts varied significantly when compared to group II and III at 2^nd week of T-2 toxin feeding. After 8^th week of T-2 toxin feeding the per cent neutrophils counts differed significantly amongst the groups as compared to that of control in dose and duration dependent manner. Highest per cent was observed in group III followed by group II and group I in comparison to group IV at 12^th week of T- 2 toxin feeding. Per cent basophil counts did not vary significantly amongst all the groups. Similarly, statistically significant (P<0.05) differences were not recorded in eosinophil and monocytes counts in all the treatment as well as the control groups at all intervals. Total thrombocyte counts revealed that T-2 fed animals had significantly (P<0.05) less number of thrombocytes in a dose dependent manner at all intervals except at 2^ndweek.

The clinical signs of variable degree of dullness, weakness, lethargy, retardation in growth, rough hair coat, disinclination to move and reduced feed intake. The clinical signs exhibited by the animals were similar to those reported earlier by various workers in rats⁸,⁹, mice ¹⁰,¹¹ and in guinea pigs¹². The behavioral changes such as loss of appetite and disinclination to move were likely mediated through CNS involvement and were triggered by neurochemical changes in the brain. Inhibition of protein synthesis in liver and other tissues after exposure to T-2 led to increased brain uptake of tryptophan. This subsequently caused increased synthesis of serotonin and resulted in exaggerated characteristic behavior¹³. The acute oral dose of T-2 toxin caused sequential elevation of tryptophan, serotonin and 5- hydroxyindoleacetic acid in rat brain which contributed to feed refusal in animals suffering from T-2 toxicosis¹⁴. Feed refusal induced by T-2 toxin due to inflammation of mucosal surfaces of the gastrointestinal tract was reported by earlier worker¹⁵. However, in the present study inflammation of GI tract was not observed in T-2 treated rats which might be due to low concentration of T-2 toxin used which did not damage the epithelial cells and was in agreement with previous workers in rats¹⁶. Facial and podal dermatitis seen in 1.0 ppm toxin fed group in the present study was also described by many workers in rats¹⁶, mice¹¹,¹⁷ and poultry¹⁸. Appearance of tail, facial and podal dermatitis might be due to the irritant nature of T-2 toxin to the skin¹⁵ and might also be due to cytotoxic radiomimetic effects of T-2 toxin on actively dividing cells of skin⁵.

Dose and duration dependent decreases in body weight gains observed in present investigation in treatment group animals could be attributed to T-2 toxicosis and have also been reported in rats⁸,⁹,¹⁶, mice¹⁰,¹¹, guinea pigs¹² and poultry¹⁸,¹⁹. The reduced body weight gain in T-2 toxicated rats might be due to irritation of digestive system resulting into decrease in feed digestion and absorption and consequently decrease in body weight of toxicated rats. Other possible reasons might be anorexia/reduced feed consumption and damage to vital organs such as liver and kidneys, as observed histopathologically, affecting the synthesis of proteins at a normal rate by the damaged liver⁷ and even loss of more protein in the urine in T-2 treated rats leading to emaciation and loss of body weight. The mortality ranged from 6.25% (group I), 8.33% (group II) and 12.00% (group III) in T-2 treated animals could be attributed to fatal kidney damage and hepatotoxicity as was evidenced histologically in present study and also been observed in rats earlier⁸.

The T-2 toxicated rats exhibited anaemia, usually of microcytic and hypochromic type as evidenced by decrease in haemoglobin, TEC, MCV, MCH and PCV values in a dose dependent fashion. These observations were in agreement with the previous workers in mice¹⁰,²⁰, rats²¹ and poultry¹⁸,¹⁹. Decrease in Hb concentration could possibly be due to decreased protein synthesis in toxicated animals¹⁸, cytotoxic effect of T-2 toxin on haemopoietic progenitor cells and enhanced excretion of porphyrins due to inhibition of haemoglobin synthesis by T-2²¹. Faifer and Godoy²² reported that T-2 toxin strongly inhibited¹⁹ Fe uptake into circulating erythrocytes and transiently depleted granulocyte macrophage colony-forming cells in the bone marrow in dose dependent manner further explains the mechanism of anemia due to T-2 toxicity. Interaction of T-2 with the inner leaflet of the lipid bilayer of RBC led to invagination of cell membrane. This alteration in cell membrane of erythrocyte caused hemolysis²³. Free radical induced hemolysis due to T-2 toxicosis was generally attributed to oxygen containing free radicals which peroxidized the polyunsaturated fatty acid in the erythrocyte membrane²³. Other factors contributing to cause anaemia could be decreased nutritional intake due to anorexia and hypoproteinemia due to damage to vital organs particularly the liver⁷ as also observed in the present study.

Leucocytopenia due to lymphocytopaenia observed in T-2 treated animals in a dose dependent manner corroborated with the findings of earlier workers in different species of animals like cat, mouse, guinea pig, rat, rabbit, monkey and pig¹⁰,¹⁷,²¹,²⁴,²⁵,²⁶. In an in vivo study haematopoietic tissues were target of toxicity in several animal species such as mice, rats, cats, rabbits and guinea pigs following acute exposure to one or more doses of T- 2 toxin²⁴. The leucocytopenia due to lymphocytopenia might be attributed to reduced proliferation of lymphocytes or direct lymphocytes cytotoxicity caused by T-2 toxicosis²⁷. On the contrary, per cent neutrophils were higher in toxin groups, which may be due to the relative reduction of lymphocytes. This finding was similar to that of the observations made by earlier workers in mice¹⁰,¹⁷,²⁵. The leucocytopenia was further substantiated by the depletion of lymphoid cells in lymphoid organs like spleen, thymus and Peyer's patches recorded histopathologically in the present study and got supported from previous reports¹⁰,¹⁹. The peripheral lymphocytopenia together with lymphoid depletion in lymphoid organs might have resulted decreased in both humoral and cell mediated immune response in T-2 treated rats in the present investigation. T-2 fed animals had significantly less number of thrombocytes (thrombocytopenia) at 2 week onwards, and this was supported by the earlier workers in mice²⁵ and birds¹⁹,²⁹. Thrombocytopenia might be due to adverse effect of T- 2- toxin on proliferation and differentiation of platelet progenitor cells leading to cytotoxicity²¹.

Means bearing at least one common superscript (a,b,c,d and A,B,C,D,E,F,G,H,I,J,K,L) do not differ significantly between groups and weeks (P<0.05); Group I (0.5 ppm), Group II (0.75 ppm), Group III (1.0 ppm), Group IV (Control)

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 (P<0.05); Group I (0.5 ppm), Group II (0.75 ppm), Group III (l.0 ppm), Group IV (Control); Hb = Hemoglobin; PCV = Packed cell volume; TEC = Total erythrocyte count; MCV = Mean corpuscular volume; MCH = Mean corpuscular hemoglobin; MCHC = Mean corpuscular hemoglobin concentration; TLC = Total leucocyte count; Lymph. = Lymphocyte; Neu. = Neutrophil; Eos. = Eosinophil; Mono. = Monocyte; Baso. = Basophil, TTC = Total thrombocyte count

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