Ameliorative effects of dietary clinoptilolite on pathological changes in broiler chickens during aflatoxicosis

 

AFLATOXINS (AF), are secondary metabolites produced by certain fungi belonging to the genus Aspergillus and can occur as natural contaminants of poultry feed (Leeson et al 1995, Oguz 1997). The toxicity of AF in broiler chickens has been widely investigated by the determination of their carcinogenic, mutagenic, teratogenic (Chattopadhyay et al 1985, Sengstag et al 1996, Celik et al 2000) and growth inhibitory (Kubena et al 1993, 1998, Santurio et al 1999, Oguz and Kurtoglu 2000) effects. The biochemical-haematological (Fernandez et al 1994, Amer et al 1998, Ibrahim et al 1998, Oguz et al 2000a), immunological (Ghosh et al 1990, Gabal and Azzam 1998, Qureshi et al 1998) and pathological (Chattopadhyay et al 1985, Dafalla et al 1987, Espada et al 1992, Kiran et al 1998) toxic effects of AF have also been well described.

Aflatoxicosis in poultry causes listlessness, anorexia with lowered growth rate, poor feed utilisation, decreased weight gain, decreased egg production, increased susceptibility to environmental and microbial stresses, and increased mortality (Bailey et al 1998, Kubena et al 1998). Also associated with aflatoxicosis are important gross and histopathologic changes in major organs and these can assist in the diagnosis of toxication. The pathological signs in broilers are characterised by hepatic lesions such as enlarging, paleness, hydropic degeneration, fatty change, bile duct hyperplasia and periportal fibrosis (Chattopadhyay et al 1985, Dafalla et al 1987, Ledoux et al 1999) and kidney and spleen lesions (Bilgic and Yesildere 1992, Espada et al 1992, Marquez and Hernandez 1995). Aflatoxins can also cause impairment of the humoral and cellular immune responses and increases susceptibility to some environmental and infectious agents (Giambrone et al 1985, Gabal and Azzam 1998, Qureshi et al 1998, Ibrahim et al 2000). Atrophy of the thymus and Bursa of Fabricius (BF; Chattopadhyay et al 1985, Kiran et al 1998) and lymphocytopenia have been reported in aflatoxicosis (Oguz et al 2000a).

Producers and researchers desire to develop an effective decontamination technology dealing with this feed-borne toxin (Leeson et al 1995). Approaches used have included the physical, chemical and biological treatment of contaminated feeds and feedstuffs. A successful detoxication process must be economical, must be capable of eliminating all traces of toxin without leaving harmful residues and must not impair the nutritional quality of the commodity (Bailey et al 1998, Kubena et al 1998, Parlat et al 1999). In the last 10 years, several studies have been performed using adsorbents for detoxifying AF in contaminated feed and feedstuffs (Harvey et al 1993, Kubena et al 1993, Jindal et al 1994, Abo-Norag et al 1995, Bailey et al 1998) and other poultry species (Sjamsul et al 1990, Kubena et al 1991, Parlat et al 1999). The non-nutritive clays such as aluminosilicates (Kubena et al 1990, 1993, 1998, Ledoux et al 1999), zeolites (Scheideler 1993, Kececi et al 1998, Miazzo et al 2000), bentonites (Dale and Wyatt 1995, Ibrahim et al 1998, 2000, Santurio et al 1999, Rosa et al 2001) and clinoptilolite (CLI; Harvey et al 1993, Parlat et al 1999, Oguz and Kurtoglu 2000, Oguz et al 2000a,b) were preferred because of their high binding capacities against AF and their reducing effect on AF-absorption from the gastrointestinal tract. It has been reported that CLI was inert and non-toxic for poultry (Olver 1997, Balevi et al 1998) but had toxicologic importance (Poschl and Balas 2000). Furthermore, it has also been approved by EU-Feed-Legislation as a feed additive in pigs and poultry feed from the year 2000 onwards (Bluthgen and Schwertfeger 2000).

Most studies have used greater concentrations of AF than can naturally occur in the field condition. The AF concentrations in these experiments ranged from 2 to 5 mg kgÿ1 diet(Kubena et al 1990, 1993, 1998, Kiran et al 1998, Oguz et al 2000a, Miazzo et al 2000, Ibrahim et al 2000, Rosa et al 2001) because these high concentrations may help to elicit the toxic effects of AF and also any effects of the adsorbents would be easily seen in a shorter experimental period. Different adsorbent concentrations ranging from 5 to 30 g kgÿ1 were used in the studies above. Therefore, the purpose of the present study was to evaluate the toxic effects of AF by pathological examination; to determine the preventive effectiveness of different dietary concentrations of CLI (15 and 25 g kgÿ1 ); and to compare the efficacy of these two different dietary concentrations.

 

 

Three hundred and sixty 1-day-old unvaccinated broiler chicks (Avian strain) of both sexes were obtained from a commercial hatchery. Individually weighed chicks were divided at random into six groups. The birds were housed in electrically heated batteries under fluorescent lighting. The chicks were fed a commercial diet (maize and soybean meal diet providing 230 g protein, 13
26 MJ ME kgÿ1 ) formulated to contain National Research Council (1994) requirements of all nutrients, without added antibiotics, coccidiostats, or growth promoters. The diet and water were always available and the lighting was continuous for 21 days. The basal diet was tested for possible residual AF before feeding (Howel and Taylor 1981) and there were no detectable levels present (detection limit 1 mg kgÿ1 diet; recovery of the extraction method 95 per cent).

The experimental design consisted of six dietary treatments. 1) CONT: Basal diet; 2) AF: basal diet plus 2
5 mg total aflatoxin (AF; the composition given below) kgÿ1 diet; 3) CLI(1
5 per cent): basal diet plus 15 g CLI kgÿ1 diet; 4) AF ? CLI (1
5 per cent): basal diet plus 2
5 mg AF plus 15 g CLI kgÿ1 diet; 5) CLI (2
5 per cent): basal diet plus 25 g CLI kgÿ1 diet; 6) AF ? CLI(2
5 per cent): basal diet plus 2
5 mg AF plus 25 g CLI kgÿ1 diet. Clinoptilolite (CLI/ NUT-1000TM), which is a member of heulandite-stilbite group, was provided from west region of Turkey and its chemical formula is KNa2Ca2 (Si29AL7) O72
32H2O.

Aflatoxin was produced from Aspergillus parasiticus NRRL 2999 culture (USDA, Agricultural Research Service, Peoria, IL, USA) via fermentation of rice by the method of Shotwell et al (1966) with minor modifications by Oguz (1997). Successfully fermented rice was then steamed to kill the fungus, dried and ground to a fine powder. The AF content in rice powder was analysed by the method of Shotwell et al (1966) and measured on thin-layer chromatography (TLC)-fluorometric densitometer (Perkin Elmer MPF 43-A) on the TLC spots at 365 excitation and 425 emission wavelengths. The AF within the rice powder consisted of 76
40 per cent AFB1, 16
12 per cent AFB2, 6
01 per cent AFG1 and 1
47 per cent AFG2 based on total AF in the ground rice powder (detection limit 1 mg AF kgÿ1 rice powder; recovery of the extraction method 92 per cent). The rice powder was incorporated into the basal diet to provide the required amount of 2
5 mg AF kgÿ1 feed.

When the chicks reached 3 weeks of age, the feeding trial wasterminated and 10 broilersfrom each treatment were selected at random and killed for pathological examination. [Other chicks in the groups were used for serum biochemical±haematological and performance investigations, and findings were published previously (Oguz and Kurtoglu 2000, Oguz et al 2000a)]. Selected animals were weighed before being killed. A detailed necroscopy was then conducted. The liver, kidney, spleen, thymus and bursa of Fabricius were removed and weighed. The relative organ weights (g of organ 100 gÿ1 of live body weight) were calculated. Tissue samples from these organs were collected in 10 per cent neutral buffered formalin. After fixation, samples were dehydratedinalcohol, clearedinxylene andembeddedin paraffin wax. Sections were cut at 5 mm and stained with haematoxylin and eosin. Some sections were also stained van Gieson and Periodic Acid Shiff reagent. Buffered formalin-fixed liver blocks were sectioned by cryostat at 10 to 12 mm and stained for lipids using Sudan black.

The differences among the relative organ weights were analysed by Duncan's multiple range test. The differences among the pathological changes were also determined by chi-quare test (SPSS 1988). Statements of statistical significance are based on P < 0
05.

The results presented in Tables 1 and 2 show the effects of dietary treatments on relative organ weights and gross-histopathological changes. The severity of the changes were classified from slight to severe. Feeding AF alone caused significant increases in relative weights of liver and kidney (Table 1). Grossly, the livers of chicks that had consumed AF were enlarged, pale yellow, friable and with rounded margins, while AF plus CLI (1
5 and 2
5 per cent) groups livers showed relatively less severe hepatic lesions (Figs 1, 2). The kidneys in some cases from AF-fed chicks were pale, enlarged and mottled in appearance due to congestion. Aflatoxin also caused reductions in the size of the thymus (Fig 3) and BF (in eight cases), and petechial haemorrhage in thigh muscles and thymuses.

Microscopically, in chickens in the AF-fed group the livers showed diffuse, moderate-to-marked hydropic degeneration. This degeneration was more pronounced in centrilobular areas (Fig 4A). In severe cases, some hepatocytes had pycnotic nuclei, and were so swollen that several cells had ruptured. The sinusoids were shrunken or completely plugged due to swollen hepatocytes, and there were few or no erythrocytes in the central vein. Moreover, the livers of some chicks fed the AF diet alone had moderate to severe cytoplasmic vacuolation of periportal hepatocytes, indicating fatty change. This condition was confirmed with Sudan black stain. In nine cases, the livers of chicks consuming AF alone showed dissociation and/or acinar arrangement of hepatocytes, surrounded by a thin stroma, suggesting hepatocellular regeneration (Fig 4B). Periportal fibrosis (Fig 5A), bile-duct hyperplasia (Fig 5B), nodular lymphoid cell accumulation, sometimes heterophil and mononuclear cell infiltrations or both in portal areas were also seen in this group.

Chicks fed AF alone also had significant renal disease with increases in the amount of mesengial cells, thickening in glomerular basement membrane, adhesions between glomeruli and bowman capsules (Fig 6), hydropic degeneration and/or cloudy swelling in tubular epithelium and focal mononuclear cell infiltrations in interstitial areas. Follicular depletion in the spleen of AF fed chicks was also observed. Severe depletion of lymphoid cells was seen in the thymuses of chicks fed AF alone. In some cases, follicular necrosis and depletion were so pronounced that the cortex remained as a thin layer at the periphery of follicles and the medulla relatively expanded (Fig 7); haemorrhage in both medulla and cortex of thymuses was occasionally seen in AF-fed group. There was also severe depletion of lymphoid cells in the BF from AF fed chicks and some of the remaining lympocytes had pyknotic or karyorrhectic nuclei (Fig 8).

 

The addition of CLI (1
5 and 2
5 per cent) to AF-containing diet (Group 4 and 6) partially (completely in some cases) decreased both the incidence of affected broilers and the severity of lesions in the organs of chicks (Table 2, Figs 1, 2, 7B, 8C). The additions of CLI to the AF-free diet (Group 3 and 5) did not cause any significant gross and histologic changes in chicks compared to controls (Table 2).

Aflatoxins are important to the poultry industry because of their frequent occurrence in feedstuffs which produces severe economic losses and health problems in poultry (Leeson et al 1995, Santurio et al 1999). Diagnosis is rather difficult and medical treatment may be almost impossible. The gross and histologic investigations in the previous studies showed that AF affected the organs belonging to the haematopoietic, immune and reticulo-endothelial systems (Kiran et al 1998, Kubena et al 1998, Qureshi et al 1998). In this study, the toxic effects of AF on the pathological base and the ameliorative efficacy of dietary adsorbent (CLI) on the detrimental effects of AF were investigated.

Liver, kidney and the immune system organs are considered to be target organs for AF and these are primarily affected in aflatoxicosis cases. In this study, significant gross and histopathological changes/lesions (indicated in Table 2 and Results section) were seen in liver, kidney, spleen, thymus and BF from the chicks consumed AF-alone diet (all Tables and Figs). These changes strongly confirm the results of previous AFstudies (Chattopadhyay et al 1985, Dafalla et al 1987, Bilgic and Yesildere 1992, Espada et al 1992, Fernandez et al 1994, Ledoux et al 1999). The increase in the relative weight of livers induced by AF is attributed to an accumulation of lipid in the liver, which produces the characteristic, enlarged, friable, fatty livers associated with aflatoxicosis in broilers (Kubena et al 1993, Ibrahim et al 1998). The hepatotoxic effects of AF caused impairments in protein, carbohydrate and lipid metabolism, decreases in serum protein, uric acid and cholesterol levels (Kubena et al 1993, 1998, Ledoux et al 1999), and inhibition of haematopoiesis (Oguz et al 2000a). The AF-related-impairments in coagulation mechanisms and capillary endothelial fragility increase the tendency to haemorrhage in the body (Dafalla et al 1987, Kiran et al 1998). The haemorrhages seen (in fivecases) in legs and breast muscles and thymuses from the chicks fed AF alone in this study may be attributable to these causes. The AF-related kidney lesions also cause important failure in the calcium, phosphorus and uric acid metabolism, and relatively the bone and egg shell quality are affected in poultry (Glahn et al 1990, 1991). In the present study, kidney lesions induced by AF were observed in seven cases

Thymuses and BF have responsibility in both cellular and humoral immunity in broilers and these organs must be functional throughout their life (Qureshi et al 1998). Regression of thymus and bursal developments would appear in the poor immune response in chicks and may cause failure in vaccination. In this study, both gross and histopathologic changes such as atrophy and lymphoid cell depletion were observed in thymuses and BF in chicks fed an AF diet alone in most cases (Table 2, Figs 7, 8). Six chicks fed AF alone showed slight cell depletion in the spleen. These spleen lesions also support the immunotoxic and haematotoxic effect of AF (Gabal and Azzam 1998, Ibrahim et al 2000). It was concluded that, the AF exposure could affect the cellular and humoral immunity of chicks and AF would impair immunological performance and increase susceptibility of animals to environmental and infectious agents.

Removing AF from contaminated feed and feedstuffs remains a major problem and there is a great demand for effective decontamination technology (Abo-Norag et al 1995, Bailey et al 1998). An encouraging approach to the problem has been to use non-nutritive and inert adsorbents in the diet to bind AF and reduce the absorption of AF from the gastrointestinal tract. These compounds must not be absorbed from the gastrointestinal tract and must have the ability to bind physically with chemical substances, precluding their absorption (Santurio et al 1999, Miazzo et al 2000). In this study, CLI was selected as the adsorbent for the diminution of the AF absorption from the gastrointestinal tract and the amelioration of the toxic effects of AF in broilers. Clinoptilolite has been reported as inert and non-toxic matter for poultry (Olver 1997) and approved by EU 2000 onwards (Bluthgen and Schwertfeger 2000). The findings in this study have supported these reports, because the additions of CLI (1
5 and 2
5 per cent) to the AF-free diet (Group 3 and 5) did not cause any detrimental effects on investigated parameters in this study (Table 2).

The additions of CLI (1
5 and 2
5 per cent) to the AFcontaining diet (Group 4 and 6) partially reduced both the number of affected broilers and the severity of lesions in the organs examined (Tables 1, 2, Figs 1B, 2B). In this study, CLI acted as a sequestering agent against AF in feeds through its adsorption and therefore reducing the AF bio-availability in the gastrointestinal tract. The CLI±AF interaction involves the formation of complex by the B-carbonyl system of the AF with free radicals of the adsorbents (Bailey et al 1998, Santurio et al 1999). In these current findings, it can be noted that no significant differences were found between the preventive efficacy of the two doses of CLI (1
5 to 2
5 per cent). Previous studies have reported that the addition of CLI (1
5 per cent) to the AF (2
5 mg kgÿ1 )-containing diet significantly reduced the growth inhibitory effects (Oguz and Kurtoglu 2000) and biochemical±haematological toxic effects (Oguz et al 2000a) of AF in broiler chicks fed for 21 days. In a long-term trial, Oguz et al (2000b) reported an intermediate reduction in the adverse effects of low levels AF (100 mg kgÿ1 ) on toxication in broilers by dietary CLI (1
5 per cent) for 42 days. These studies reported that the lower levels of CLI (1
5 per cent) in feed was more effective for preventive efficacy than higher levels of CLI (2
5 per cent) in the parameters investigated. CLI also provided significant improvements on performances of Japanese quail by the addition of CLI to the AF (2 mg kgÿ1 )-containing diet for 35 days (Parlat et al 1999). However, Harvey et al (1993) found no beneficial effect on broiler chicks of adding CLI (0
5 per cent) to an AF (3
5 mg kgÿ1 ) containing diet for 21 days. The reason for these conflicting results may be attributable to differences in type, dose, physical characteristics of CLI, concentration of AF in the diet or broiler strain.

These results clearly demonstrate that the simultaneous addition of CLI to the AF-containing diet provided a moderate amelioration in AF toxicity. These improvements should contribute to a solution of the AF problem in broiler chickens when adsorbents are used in conjunction with other AF prevention strategies.

 

 

The authors gratefully acknowledge `The Scientific and Technical Research Council of Turkey (TUBITAK)', whose Veterinary Medicine and Animal Husbandry Research Grand Committee funded this project. Project no: VHAG-1437.

 

 

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