INEFFECTIVENESS OF DIFFERENT ADSORBENTS IN ALLEVIATION OF ORAL LESIONS INDUCED BY FEEDING T-2 TOXIN IN BROILER CHICKENS

One hundred and forty one-day-old-male broiler chicks (Ross), were divided into 7 groups (20 chicks/group) and fed T-2 toxin alone or with five different types of adsorbents; Montmorillonite 0.5%, Vermiculite 0.5%, Pigacine 0.5%, Gezilgure 0.5%; and the resin Perlite 0.5%. Gross and microscopic examinations reveal that all adsorbents used were unable to alleviate the oral lesions induced by T-2 toxin in broiler chickens.


INTRODUCTION
Trichothecene mycotoxins are produced by Fusarium and its perithecial stages (1).T-2 toxin, one member of group A trichothecenes, was first isolated from fusarium tricictum strain T-2 by Bamburg (2) and Bamburg et al., (3).Feeding T-2 toxin to broilers cause feed refusal, impaired growth and whole body pathology including caustic injury to skin and alimentary mucosa; radiomimetic injury to bone marrow, lymphoid tissues, gastrointestinal tract, and hepatitis; and thyroid alteration(4).T-2 toxin causes erosive and exudative injury to the oral mucosa of poultry fed toxin-appended diets (5, 6, and 7)).These oral lesions were characterized by the presence of focal, yellow oral plaques that progress to yellow gray, raised accumulations of exudates with underlying ulcers located near major salivary duct openings on the palate, tongue, and buccal floor.Thick crusts accumulate along the interior margin of the beak.Oral histology confirms mucosal necrosis and ulceration, sub mucosal granulation tissue and inflammatory cells, crusts of exudates, bacterial colonies, and feed components.(5; 8; 9; 10; 11; 12; 13; 14).The most applied method for protecting against mycotoxicosis is the utilization of adsorbents mixed with the feed which are supposed to bind the mycotoxins efficiently in the gastrointestinal tract, (15).Most studies related to the alleviation of mycotoxicosis by the use of adsorbents are focused on aluminosilicates, mainly zeolites and Hydrated sodium calcium aluminosilicates(HSCAS), and aluminumonsilicate-containing clays.All consisting of aluminates, silicates and some interchangeable ions, mainly alkali metal and alkaline earth metal ions (16).Clay minerals are primarily layered silicates with the common chemical formula[Sio2O 2 5] xy, e.g.Bentonit, vermiculite, Kaolin, Pigacin, Gezilgure.HSCAS contain calcium ions and protons which are exchanged against the naturally occurring sodium ions.They are a type of montmorillonite belonging to phyllosilicates which are composed of layers of aluminum and silicon connected in a 1:1 or 2:1 arrangements.Polymers, an ion exchange mycotoxin adsorbent resins were also tried to be used in vitro studies for adsorption of certain mycotoxins (15,17).As a general aluminosilicates are the preferred adsorbents, followed by activated charcoal and special polymers(16).The aim of the present study was to evaluate different adsorbents; Montmorillonite (HSCAS); new aluminosilicte-containig clays (Vermiculite, Pigacin, Gezilgure); and new polymer resin (Perlite), in an attempt to alleviate necrotic oral lesions induced by feeding T-2 toxin to broiler chickens.
The experimental ration was checked to contain no detectable levels of aflatoxins, ochratoxins, zearalenone, and T-2 toxin by the method reported by Coker et al (1984) (19).Purified crystalline T-2 toxin was produced by culturing F. tricinctum NRRL 3299 according to the method reported by Burmeister et al. (20).The crystalline toxin was dissolved in acetone, added to experimental diets, and mixed to homogeneity by means of a twin-shell blender.Homogeneity was tested by analyzing the presence of T-2 toxin in each diet by thin-layer chromatography.The chemical composition of Aluminum silicate feed additives are presented in table 1. Chickens were kept in batteries in a thermo regulated room with continuous light; feed and water were provided ad libitum.On 21 day of age heads were removed from all chicks and visually scored for oral lesions using a four -point scoring system ranging from 1 to 4 (5) by the same individual without knowledge the treatment groups.A lesion score of 1 indicated no visible lesions; a score of 2 was seen as one or two mouth lesions clearly visible on either the lower or upper mandible; a score of 3 was seen as more than one or two mouth lesions clearly visible on either the lower or upper mandible and back of the tongue ; a lesion score of 4 was seen as large lesions occurring at several sites within the mouth, principally on the upper and lower mandibles, the corners of the mouth, and the back of the tongue.Histopathological study of oral lesions was performed by fixing affected portions in 10% neutral buffered formalin.Fixed tissues were trimmed, embedded in paraffin, sectioned at 4 µm, and stained with hematoxylin and eosin stain.Tissue samples from treatments show oral lesions were examined microscopically.Data (pen means) for all response variables in each treatment were subjected on ANOVA using the SAS software (SAS 2000) general Linear Models procedure for factorial ANOVA.Treatment means were ranked by Duncan's multiple range test (SAS 2000).All statements of significance are based on the 0.05 level of probability (21).

Oral lesion scores:
The oral lesion scores in all treatment groups fed purified T-2 toxin with five different aluminum silicate feed additives show no significant difference in developing oral lesions when compared with group of chicks fed T-2 toxin alone, but all were significantly (p<0.05)differ from control group fed no toxin or additives(Table 1). 2 Lesions were scored on a scale from 0-4, with a value of 4, representing the more sever lesions.
Gross lesions: Oral lesions were reported in all groups fed T-2 toxin alone or with different feed additives and were characteristic of T-2 toxicosis which first appeared when the chicks were about 1 week old, as raised ceasous yellowwhite plaques.By 2-weeks, the lesions increased in size and invaded the lingual papillae at the root of the tongue, margin of the beak, mucosa of the hard palate and angle of the mouth.By three weeks, the size of the lesions increased to more extent that of the 2 nd week.These lesions were characterized by raised accumulation of exudates with underlying ulcers located near major duct opening of the palate, tongue, and buccal floor.(Figure 1      Microscopical findings: Histopathological results 0f 21-day-old experimentally fed broiler chickens 8 ppm T-2 toxin showed intense inflammation and localized necrosis in the oral cavity.The outer layer of the lesion consisted of sloughing fibrous material with bacterial colonies, while the underlying tissue was heavily infiltrated with granular leukocytes.Hyperplasia of the main salivary glands in the oral cavity was also evident (Figure 6) .Montmorillonite, hydrated sodium calcium aluminosilicate (HSCAS), when added at a rate of 0.5% to T-2 toxin contaminated diet was ineffective in counteracting adverse effect of T-2 toxin inducing oral lesion.Lesions were characterized by Sloughing upper fibrous material which was heavily contaminated with bacterial aggregations, while underlying tissue was infiltrated with granular leukocytes (Figure 7).Increase in the thickening of the mucous membrane was noticed as a result of an increase in the amount of exudates, granulocytes and subcutaneous edema (Figure 9).The addition of 0.5% Gezilgure, the third mineral clay used, to T-2 toxin contaminated diet, was also failed to relief the degenerative effect in broiler buccal mucosa induced by the toxin.Oral histopathology confirms mucosal necrosis which varied in shape and size and had random distribution.The lesions were consisted predominantly of lower hyperplasic epithelium, layer of inflammatory cells, crusts of exudates, bacterial colonies and feed component (Figure 10).ppm(23,24); 8 ppm(6, 25,26,27).Gross and microscopic oral lesions induced here were resemble those reported early by Wyatt et al (12,28) in broilers fed diets containing 1ppm T-2 toxin and more.These oral lesions were also reported in broilers fed purified T-2 toxin (produced and extracted from F. tricictum) at a rate of 4-16 ppm for 3 weeks, and some microorganisms were isolated from these lesions (10).The erosive and exudative injury to the broiler oral mucosa, and the increase in the bacterial clumps in the sloughed outer fibrous layers appear to bear some resemblance to those described for Alimentry Toxic Aleukia in humans (12; 29).In humans, necrotic lesions were reported in gums, buccal mucosa, throat, larynx and vocal cords, contaminated with a variety of avirulent bacteria.The necrotic areas are an excellent medium for bacterial infection due to lowered résistance caused by damage to the haemopoitic and reticuloendothelial systems (30).These bacterial infections induced by T-2 causes an unpleasant odor from the mouth due to the enzymatic activity of the bacteria on proteins.In the results here, it is interesting to note that in all groups fed T-2 toxin amended with all used adsorbents, there was an increasing tendency to bacterial colonies aggregations in the necrotized, sloughed superficial layer of oral lesions.The exacerbating effect of these silica containing adsorbents (montmorillonite, 33.00%; Vermiculite, 35.12%; pigacine, 61.09%; gezilgure, 88.3%; perlite, 73.35%) in predisposing bacterial colonization may be due to the specific silica-macrophage cytotoxic interaction (31).This condition was also confirmed and reported when broiler chickens fed siliceous Ninivite (6).The increase in bacterial infection could be attributed to T-2 toxin itself in leucopenia and anemia induction (6, 7).The dramatic consequences of T-2 toxin feeding to broiler chicks could be attributed to large extent to its caustic property.The mucosal necrosis caused by Y-2 toxin is very painful to the affected birds, thus feed consumption decreases.Subsequently weight gain and feed conversion are reduced (7).
The most important feature of the adsorption is the physical structure of the adsorbent, i.e. the total charge and charge distribution, the size of the pores and the accessible surface area.On the other hand, the properties of the adsorbate molecules, the mycotoxins, like polarity, solubility, size, shape and -in case of ionized compounds-charge distribution and dissociation constants play a significant role, too.Therefore, the efficacy of every adsorption process has to be investigated in regard to the particular properties of the adsorbate (16).The applicability of aluminosilicates for the adsorption of mycotoxins has been studied for more than 20 years (32).All these studies refer to inability of aluminosilicates to counteract the negative effect of T-2 toxin and other trichothecenes (16).Of these studies which confirm the present results those used hydrated calcium sodium aluminosilicate (HSCAS) at a rate of 0.25%; 0.37%; 0.5%; and 0.8%, and showed that there was no effect against toxicity induced by 8 ppm T-2 toxin in broiler chickens (25,26).No references are available in using clay minerals or resin polymers for counteracting T-2 toxicosis in broilers.So , it is the first time that these clay minerals and resin polymers were tested to elucidate their effectiveness in alleviating oral lesions induced by feeding purified T-2 toxin to broiler chickens.T-2 toxin has a toxic specific group on position (12, 13) responsible for the molecules negative impact on performance and health of animals.Enzymes (de-epoxydases and esterase) are at the present the only way to detoxify T-2 toxin and reduce its toxicity by converting the toxin to nontoxic product.
Figure 1: Beak and palate ulceration and crusting in broiler chickens following 21 days consumption T-2 toxin (8 ppm), characterized by raised accumulations of exudates with underlying ulcers located near major salivary duct openings on the palate.

Figure 2 :
Figure 2: Beak and palate ulceration and crusting in broiler chickens following 21 days consumption 8 ppm T-2 toxin and 0.5% montmorillonite, characterized by raised accumulations of exudates with underlying ulcers located near major salivary duct openings on the palate.

Figure 3 :
Figure 3: Beak and palate ulceration and crusting in broiler chickens following 21 days consumption 8 ppm T-2 toxin and 0.5% vermicullkite, characterized by raised accumulations of exudates with underlying ulcers located near major salivary duct openings on the palate.

Figure 4 :
Figure 4: Beak and palate ulceration and crusting in broiler chickens following 21 days consumption 8 ppm T-2 toxin and 0.5% pegacine, characterized by raised accumulations of exudates with underlying ulcers located near major salivary duct openings on the palate.

Figure 5 :
Figure 5: Beak and palate ulceration and crusting in broiler chickens following 21 days consumption 8 ppm T-2 toxin and 0.5% gezilgure, characterized by raised accumulations of exudates with underlying ulcers located near major salivary duct openings on the palate.

Figure 6 :
Figure 6: Beak and palate ulceration and crusting in broiler chickens following 21 days consumption 8 ppm T-2 toxin and 0.5% perlite, characterized by raised accumulations of exudates with underlying ulcers located near major salivary duct openings on the palate.

Figure 6 :
Figure 6: Photomicrograph of oral lesions of 21-day-old broiler fed purified 8 ppm T-2 toxin.(A) Oral mucous membrane show intense inflammation and localized necrosis, sloughing of upper fibrous material contained bacterial aggregations, while underlying tissue was heavily infiltrated with granular leukocytes; hyperplasia of the epithelial layer and salivary glands is also evident.H&E.X36 ;( B) Higher magnification of A, H&E.X160; (C) Hyperplasia of oral salivary gland in(A).H&E.X80.

Figure 7 :
Figure 7: Photomicrograph of oral lesions of 21-day-old broiler fed purified 8 ppm T-2 toxin with 0.5%.montmorillonite.(A) Oral mucous membrane showing sloughed upper fibrous material containing bacterial aggregations, while underlying tissue was heavily infiltrated with granular leukocytes .H & E. X 160 .(B) Higher magnification of (A), showing clumps of bacteria.H & E. X 700

Figure 8 :
Figure 8: Photomicrograph of the mucous membrane of 21-day-old broiler fed purified 8 ppm T-2 toxin with 0.5% vermiculite,(A) showing fibrinosuppurative inflammation and bacterial colonies in the upper layer.The underlying tissue shows inflammatory response mainly of heterophils and hyperplasia of oral salivary glands.H &E X 36.(B).Higher magnification of (A), showing fibrinosuppurative inflammation and bacterial colonies in the upper layer of oral mucous membrane.H&E.X220.

Figure 10 :
Figure 10: Photomicrograph of the mucous membrane of the buccal cavity of 21day-old broiler fed purified 8 ppm T-2 toxin with Gezilgure,(A) showing hyperplasic epithelium, layer of inflammatory cells, crusts of exudates, bacterial colonies.H&E.X36.(B) Higher magnification of (A), show cellular detritus and clumps of bacteria and feed components.H&E.X500

Table 1 :
Effect of T-2 toxin alone and T-2 toxin with feed additives on broiler oral lesions at 21days of age 1 .
*Values within column with no common subscripts differ significantly (p<0.05) 1 Values = X±SEM of two groups of 10 broilers.