HEPATIC LESIONS IN LAYING HENS CHRONICALLY EXPOSED TO RATIONS CONTAINING DIFFERENT LEVELS OF AFLATOXIN B1ABSTRACT
The aim of the present work was to study liver changes induced by chronic exposure of laying hens to aflatoxin B1 (AFB ), in view of the paucity of information on the effects of the purified toxin. To that end, four experimental groups were defined, each containing 4 commercial white laying hens (Babcock), which were fed rations containing AFBI at various concentrations (O [control], l 00 µg/kg, 300 µg/kg and 500 µg/ kg) during 60 days. After this period, the birds were fed aflatoxin-free rations for 30 days and subsequently necropsied. The livers of laying hens exposed to AFB1 appeared congested and with signs of degeneration. Histopathology revealed hepatic cell vacuolation with fatty infiltration in all the groups, which was, however, more marked in samples of hens receiving AFB1 and was maximal upon exposure to 500 µg/kg of the mycotoxin. Biliary duct hyperplasia was detected only in birds exposed to 300 and 500 µg/kg of AFB1• The sarne was observed with respect to trabecular disorder, which was more evident in the group treated with the highest dose of AFB1 • The results showed that chronic exposure to concentrations of purified AFBI above 300 µg/kg can induce significant hepatic lesions in young laying hens.
KEY WORDS:
Aflatoxin B1; toxicity; histopathology; laying hens.
RESUMO
O presente trabalho teve o objetivo de estudar as alterações hepáticas induzidas pela exposição crônica de poedeiras à aflatoxina B1 (AFB ), uma vez que a literatura é escassa de informações quanto aos efeitos da toxina pura. Deste modo, foram constituídos 4 grupos formados, cada um, por 4 poedeiras brancas de linhagem comercial (Babcock), as quais foram alimentadas com rações contendo AFB1 nas concentrações de O (controle), 100 µg/kg, 300 µg/kg e 500 µg/ kg, durante 60 dias. Após este período, as aves foram mantidas com ração isenta de toxinas por um período adicional de 30 dias, época em que se realizou a necropsia dos animais. Os fígados das aves alimentadas com ração contaminada apresentaram-se congestos e com sinais de degeneração. Observou-se a ocorrência de degeneração vacuolar microgoticular nos fígados de todos os grupos, com maior intensidade nos que receberam ração contaminada com AFB1 • A hiperplasia de duetos biliares foi constatada apenas nos grupos tratados com 300 e 500 µg/kg. O mesmo foi observado com relação ao desarranjo trabecular, mostrando-se mais evidente no grupo alimentado com 500 µg/kg. Os resultados demonstraram que concentrações de AFBI acima de 300 µg/ kg podem determinar lesões hepáticas significativas em poedeiras jovens, sob condições de exposição prolongada.
PALAVRAS CHAVE:
Aflatoxina B1; toxicidade; alterações histopatológicas; poedeiras.
INTRODUCTION
Aflatoxins are secondary metabolites produced by certain molds of the genus Aspergillus, particularly A. flavus and A. parasiticus, which cause a wide range of toxic effects in laboratory and domestic animais (Chu, 1991 ). Aspergillus spp. can grow in a variety of substrates; peanut, corn and other common ingredients of poultry feeds are rather prone to contamination with aflatoxins (Leeson et al., 1995).
Eighteen different compounds have been identified yet only aflatoxins B1 (AFB ), B2 (AFB2 ) , G1 (AFG) and G2 (AFG2 ) have been detected as natural contaminants of feeds and feedstuffs (Coulombe, 1991). These mycotoxins have closely related chemical structures but different biological activities. AFB1 shows the highest toxicity in most experimental models, while the acute toxicities of AFB2 , AFG1 and AFG2 are about 50, 20 and 10% that of AFB1 , respectively (Leeson et al., 1995).
Aflatoxins may induce acute or chronic effects, depending on the amount ingested by the animal and the duration of exposure to the toxin. Acute toxicity is clinically characterized by depression, anorexia, icterus, hemorrhages and death (Osweiller, 1990). The liver is the main target organ and the major histologic lesions include periportal necrosis and bile duct hyperplasia (Espada et al., 1992). Chronic aflatoxicosis usually results from regular low-level dietary intake of aflatoxins. In this case the most important toxic effects are hepatocellular carcinomas, bile duct hyperplasia and hepatic steatosis (McLean & Dutton, 1995).
Susceptibility to aflatoxins can vary considerably between different species. The doseresponse relationship, however, is markedly altered by intra-species factors, such as age, sex, breed and composition of diets (lqbal et al., 1983). In general, females tend to be more resistant than males, while young animais are considered the most susceptible to aflatoxicosis. Some studies suggest that, among domestic fowls, laying hens are the most resistant (Hamilton & Garlich, 1971; Arafa et al., 1981 ). According to Pier (1992), the DL50 value for 1-week-old laying hens is 6.5 mg/ kg of body weight (b.w.) after a single dose of aflatoxin.
Chronic intake of aflatoxins in feeds is the most common way of exposure of domestic animais under natural conditions. In this case, diagnosis of intoxication is particularly difficult because of the mildness of evident symptoms (Leeson et al., 1995). Some studies have revealed that leveis of aflatoxins in feed up to 500 µg/kg apparently do not induce clinical signs in adult laying hens and do not have any significant effect on egg production (lqbal et al., 1983; Washburn et al., 1985; Sudhakar, 1990).
The experimental data available on aflatoxin effects in chickens relates to naturally contaminated substrates or clean cereal grains inoculated with aflatoxigenic strains of A. flavus. The generic term aflatoxin is often used to express the content of toxin in contaminated feeds, without specifying which of the four naturally occurring aflatoxins is present (Leeson et al., 1995). However, aflatoxins differ in their toxigenic potentials and, therefore, it is particularly important to undertake studies on the specific effects of each aflatoxin, especially AFB1 , in domestic animais. In the present work, a descriptive histopathological study of aflatoxininduced hepatotoxicity was conducted on young laying hens chronically exposed to feed contaminated with low leveis of purified AFB1 •
MATERIAL AND METHODS
Sixteen commercial laying hens (Babcock) were randomly divided into 4 experimental groups (4 birds each) and placed in individual cages, which were kept in the poultry facilities of the Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo, in Pirassununga, state of São Paulo, Brazil.
Initially, the hens were fed with commercial feed for laying hens, prepared according to specifications of the National Research Council (1994), during a period of 4 weeks. The starter feed was tested for any possible residual aflatoxin, following the procedures of the CB method of analysis (Scott, 1990). When the hens were 22-week-old, the aflatoxinfree feed was replaced by rations containing AFB1 at leveis of O ( control), 100, 300 and 500 µg/kg.
The AFB1 used in this study was prepared by the Laboratory ofMycotoxins, Microbiology Department, Universi,ty of São Paulo, Brazil, using rice media cultures of A. flavus (Ross et al., 1990). After inc. bation and quantitatfon, pure AFB1 extracts were dissolved in sterile maize oil (Almeida et al., 1996) and added to clean feeds in small volumes, so as to btain the. required levels of mycotoxin. Final mixtures were homogenized in a horizontal mixer; (Marconi), in lots of approximately 30 kg each.
The prepared rations were given ad libitum for 8 weeks and then replaced by aflatoxin-free feeds for an additional period of 4 weeks. Hens were killed by decapitation and a detailed necropsy (Vasconcelos, 1988) was conducted. Liver, spleen and gizzard samples were collected in 10% neutral buffered formalin. Tissue sections 5 µm thick were stained with haematoxilin and eosin and used for histological evaluation.
Liver of laying hen exposed to rations containing 300 µg/kg of AFB1 for 60 days and subsequently fed aflatoxin-free rations for 30 days. Note hyperplasia of bile ducts and vacuolation of hepatocytes (X400).
RESULTS AND DISCUSSION
Gross pathology occurred only in the gizzard and liver ofhens exposed to AFB1 • Nodulesand apparent ulcerations of the mucosa were observed in the former organ whereas the latter appeared congested and with a slight yellowish discoloration.
Histopathology showed that the liver was the most affected organ; bile duct proliferation and trabecular disorder were found in liver samples of hens exposed to AFB1 at concentrations above 300 µg/kg (Fig. 1). Hepatic cell vacuolation with fatty infiltration was observed in liver samples of all the experimental groups, including controls. This feature might be related to physiological steatosis, which is reported to occur in practically all laying fowls under natural coriditions. (Randall & Reece, 1996). On the other hand, the intensity of such changes became more marked with increasing AFB1 levels and was maximal in hens exposed to 500 µg/kg of this mycotoxin (Fig. 2), indicating a dose-dependent response. Our results contrast with data presented by Arafa et al.{1981), who did not observe detectable hepatic lesions in 1- to 21-day-old White Leghom chickens after exposure of the birds to rations with AFB1 - equiv alent levels of 700 to 2100 µg/kg.
Nuclear degeneration (karyolysis) was also occasionally observed in liver cells of the birds treated with rations containing 500 µg/kg of AFB1 , indicating a severe intoxication, which persisted even 30 days after withdrawal of treatment.
Liver of laying hen exposed to rations containing 500 µg/kg of AFB1 for 60 days and subsequently fed aflatoxin-free rations for 30 days. Intense vacuolar degeneration of hepatic cells and trabecular disorder can be observed (X400).
Our data are in agreement with the results obtained by Camaghan et al. (1966), who observed enlarged and friable livers with a bituminous coloration and a reticulated appearance in day-old chicks that had been fed with rations containing 1500 µg/kg of AFB 1- equivalent for 8 weeks. Microscopically, the authors detected intense vacuolation of hepatocytes and proliferation of the biliary epithelium 86 hours after administration of contaminated feed.
Espada et al. (1992), working with day-old broiler chickens, observed vacuolation of hepatocytes after oral administration of AFBI at doses ranging from 0.2 to 3.0 µg/kg b.w./day during 21 consecutive days. The lesions were more intense with increasing concentrations of the aflatoxin and persisted even 10 days after withdrawal of treatment. In the present study, the daily mean consumption of rations was used to estimate the AFB 1 mean doses administered to each experimental group, namely: 6.6 µg/kg b.w./ day (100 µg/kg), 21.5 µg/kg b.w./day (300µg/kg) and 35.6 µg/kg b.w./day (500 µg/kg). Since our doses are considerably higher than the ones used by Espada et al. ( 1992), our results indicate a greater resistance of laying hens to aflatoxicosis as compared with broilers and corroborate findings previously reported (Hamilton & Garlich, 1971; Arafa et al., 1981; Pier, 1992; Leeson et al., 1995).
Histopathology of the gizzard only revealed small inflammatory foci that might have resulted from some possible friction between the organ and rough foodstuffs normally ingested by the birds. No detectable changes were observed in the spleen samples.
The results of the present work showed that chronic exposure of AFBI concentrations above 300 µg/kg may induce hepatic lesions in young laying hens. The changes observed hence constitute an important indicator of chronic intoxication by aflatoxins in the poultry industry.
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