Research in Veterinary Science 1992, 53, 275-279
Pathological lesions following an experimental intoxication with aflatoxin B1 in broiler chickens Y. ESPADA, M. DOMINGO, J. GOMEZ, M. A. CALVO, Department of Veterinary Medicine.
Faculty of Veterinary Science, Universitat Aut6noma de Barcelona, 08193 - Bellaterra, Barcelona,
Spain
A follow-up of chickens dosed orally over 21 days with 0-2 and 3 ~tg of aflatoxin B 1 (AFB1) g-I of bodyweight daily and their subsequent recovery 10 days after withdrawal of contaminated food was conducted. Vacuolation of liver cells during the initial days of the intoxication and cellular depletion in the follicle medulla of the bursa of Fabricius were the lesions which appeared first and persisted during the recovery phase in both groups of intoxicated animals. The intensity of these lesions and their persistence was related to the dose of aflatoxin ingested. A significant reduction in the bodyweight and absolute weights of fiver, bursa of Fabricius spleen and thyroid was observed in the higher dose group. IN 1961 Allcroft et al identified aflatoxins as the causal agent of the death of 100,000 poultry in Great Britain. These are secondary toxic metabolites and were elaborated and accumulated mainly by Aspergillus flavus and Aspergillus parasiticus. The ingestion of grain contaminated with aflatoxins by animals causes an aflatoxicosis, the appearance of which depends on the animal species, strain, age, nutritional state and state of health, the quantity of aflatoxin ingested and the duration of exposure to the toxin, as well as the presence of concomitant diseases and other mycotoxins (Gimeno and Martins 1983). The clinical condition and lesions, which have been widely described, are characterised by a reduction in bodyweight (Siller and Ostler 1961), deaths with nervous seizures and opisthotonos (Dalvi 1986), vacuolation of hepatic cells (Chen et al 1985, Merkley et al 1987), bile-duct proliferation (Siller and Ostler 1961, Gimeno 1988) and periportal fibrosis (Sinha and Arora 1987).
However, the chronological study of the process may be useful to establish the time sequence of appearance of lesions, as well as the persistence of some lesions and not others, after removing the toxin, from the point of view of diagnostic interest. Materials and methods
Aflatoxin and chicks Pure aflatoxin B t(AFBl), obtained from Sigma (St Louis), was dissolved in a small volume of a 25 per cent ethanol solution and the final dose was prepared with a 0.75 per cent carboxymethyl cellulose solution. The aflatoxin was administered orally. Day-old broiler chicks (150) weighing 40 to 45 g were obtained from a local hatchery. They were divided into three groups and each bird weighed daily: group C contained 50 chicks and served as the control, group D02 containing 50 chicks received 0.2 ~tg AFBl g-1 of bodyweight each day for 21 days and group D3 containing 50 chicks received 3 ~tg AFB1 g-1 of bodyweight each day, also for 21 days. After 21 days all birds were kept 10 more days without receiving further toxin. The starter and grower feed for the experimental birds were tested for any possible residual aflatoxin, following the official method of sampling and analysis of feed and raw materials according to Spanish legislation (BOE 246, 1981). Water and feed were given ad libitum and the animals were exposed daily to 18 hours of light. From the sixth until the 30th day groups of three animals were killed by decapitation on alternate days. All animals were weighed before being
275
276
Y. Espada, M. Domingo, J. Gomez, M. A. Calvo
killed. A detailed necropsy was then conducted. The liver, spleen, bursa of Fabricius and thymus were recovered and weighed. Tissue samples from these organs and from the kidneys, lungs, cerebrum, cerebellum, proventriculus, duodenum with pancreas, myocardium and thigh muscle were collected in 10 per cent neutral buffered formalin. After fu~ation, samples were dehydrated in ethanol, cleared in xylene and embedded in paraplast at 50°C. Tissue sections 5 ~ma thick were stained with haematoxylin and eosin. Formol fixed liver blocks were sectioned by cryostat and stained for lipids using oil red O following the technique of Bancroft and Stevens (1977). Results
Macroscopic and microscopic lesions In birds of group D3, macroscopic lesions of the liver consisted of pale, yellowish discoloration of the whole organ appearing at the sixth day of intoxication and disappearing at the eighth day after withdrawal. There was enlargement of the gallbladder (from the sixth to the 14th day of intoxication), and a fine irregular surface (from the 20th day of intoxication to the sixth day after withdrawal). Microscopically, in this group, hepatic cell vacuolation with fatty infiltration was demonstrated with oil red O, surrounding the
centrilobular hepatic veins from the sixth day of intoxication. Fatty infiltration progressively extended to the whole hepatic parenchyma (by the 12th day of intoxication), and persisted nine to 10 days after withdrawal. Concomitantly with the fatty infiltration, more basophilic cells were arranged in acini around the portal areas (Fig 1). Both lesions were found in the same region and a gradual change was observed in the staining. Mild fibrosis around portal areas was only seen in some chickens on the 18th and 20th days of intoxication. Disappearance of acini ofbasophilic cells was observed on the second day after withdrawal, whereas the fatty infiltration persisted, in decreasing amount, until the end of the experiment (10 days after withdrawal). In group D02, a pale discoloration of the liver from the 16th day of intoxication to the sixth day of withdrawal was the only macroscopic lesion observed. Microscopically, fatty infiltration of liver cells became evident on the 14th day of intoxication, disappearing on the sixth day after withdrawal. In the bursa of Fabricius of group D3 a reduction in the number of cells present in the centre of the follicle was observed from the sixth day of investigation and persisted during the recovery period. Pyknosis and karyorrhexis of lymphoid cells were occasionally seen (Fig 2). A less frequent lesion of the bursa was the formation of cysts of
FIG 1 : Liver of chicken fed with 3 ~g aflatoxin B 1 g-1 bodyweight, 21 st day of intoxication. Note formation of bile ducts around a portal vessel. × 150
Aflatoxicosis in chickens
277
FIG 2: Bursa of Fabricius of chicken fed with 3 ~g aflatoxin B 1 g-1 bodyweight, 12th day of intoxication. The medullary zone of the follicles is depleted and shows pyknosis and karyorrhexis of lymphoid cells, × 150
cylindrical and cuboidal epithelium of varying sizes, located nearby or attached to the inner epithelium lining of the bursa. In group D02 cellular depletion of the bursa of Fabricius appeared on the eighth day of intoxication with recovery on the fifth day. The other lesions described in group D3 appeared only occasionally. In the kidney on the 12th day of intoxication there was evidence of an irregular dilation of some proximal tubules without evidence of cell degeneration; the lesion regressed rapidly on removal of toxin (four days). This lesion was observed only in group D3. Other microscopic lesions occasionally observed in group D3 during the intoxication period were a slight depletion of lymphoid cells in the spleen, and hyperaemia and focal haemorrhage in the medulla of the thymus. In the rest of the samples studied no lesions were observed that could be interpreted as characteristic of the process.
Absolute weight of the organs The absolute weights of the liver, spleen, thymus and bursa of Fabricius in the three groups are shown in Fig 3. A significant reduction of their weight gain throughout the intoxication period and weight gain during the recovery phase
was observed in chickens of group D3. The mean bodyweights are shown in Fig 4.
Mortality During the intoxication period 11 chicks from the D3 group died and were not included in the results because of post mortem changes. However, the same number were omitted from the other two groups at the end of the experiment. Discussion
The hepatic lesions of aflatoxicosis described as vacuolation of hepatic cells due to fatty metamorphosis are in agreement with those reported by Chen et al (1985) and Merkley et al (1987), who classified it as degenerative changes of the liver. Bile duct proliferation considered as regenerative changes of the liver was described by Siller and Ostler (1961), Bryden and Cumming (1980) and by Gimeno (1988). In this study a greater persistence of the former was observed when the toxic material was removed, although intensity was similar for both. Cell depletion in the medulla of the follicles of the bursa of Fabricius is also a characteristic lesion. Venkata et al (1988) describe this depletion of lymphocytes in chronic aflatoxicosis (12
278
Y. Espada, M. Domingo, J. Gomez, M. A. Calvo 2424 2O
Burs
Liver 2O
15
15
~z 10. 10 5.
5
2
2
0
20
10
30
-0
10
2.80 Spleen
,~
1.80 !
20
50
Days
Days Thymus
/
2.40 2.00
0-60 1.60 1.20
~o~ 0"40
0.80 0.20 0.40 0 10
20
30
Days
0
10
20
FIG 3: Absolute weights of liver, bursa of Fabricius, spleen and thymus. • = D3 (3 ,LtgAFB1) , + --- D02 (0.2 ~tg AFB1) , Aflatoxin was administered from day 0 to 21 and the weights are the mean for three birds on each occasion
weeks), although they consider that it may vary from an intense lymphocytosis to a moderate depletion of lymphoid cells; and characterise the formation of cysts in the follicles as regressive changes of the bursa. On the other hand, Giambrone et al (1985) did not observe any influence on the development of the bursa although they considered that the immune system is a very sensitive indicator of aflatoxicosis in poultry. According to Huff et al (1986), Merkley et al (1987) and Gimeno (1988), the gain of whole bodyweight and the absolute gain in weight of the organs investigated was significantly reduced in a dose-dependent relationship. Noticeably, the rate of bodyweight gain did not recover after the withdrawal of intoxication. Aflatoxin B l induced an accumulation of lipids in the liver which caused an increase in its relative weight (data not shown) and the vacuolation of
30
Days [] =
C Control group.
hepatocytes. This has also been reported by Chen et al (1985) and Merkley et al (1987), and can be explained according to Huff et al (1986) by an increase in the concentration of liver lipids, which has been demonstrated with oil red O staining. This increase may have two origins; according to Tung et al (1972) it stems from a general inhibition of lipid transport, and according to Donaldson et al (1972) from an interference in lipogenesis. The atrophy of the bursa of Fabricius, described by Merkley et al (1987) and of the thymus, reported by Gimeno (1988) and Venkata et al (1988), confirm that the immune system is a reliable indicator of aflatoxicosis. Observations on the time sequence and intensity of the lesions in relation to the dose of aflatoxin administered show that the vacuolation of hepatocytes by fatty metamorphosis and the
Aflatoxicosis in chickens 800 t Mean bodyweight
i JC:
600
400 O t,n
2O0
0 0
10
20
30
Days FIG 4: Mean bodyweight during the investigation. • = D3 (3 ~tg AFB1), + = D02 (0-2 Ixg AFSl), [ ] = C Control group. Aflatoxin was administered from day 0 to 21. The weights are the mean for three birds on each occasion
cellular depletion in the medulla of the follicles of the bursa of Fabricius were the earliest lesions and also the most persistent lesions when the toxic source was removed. It may be concluded, therefore, that these changes could be used as indicators in those cases of suspected aflatoxicosis in which the analysis of the feed ingested cannot lead to a diagnosis as it no longer contains aflatoxins. Acknowledgement This work was supported by a grant of the fruiT, Generalitat de Catalunya. References ALLCROFT, R., CARNAGHAN, R. B., SARGEANT, K. & O'KELLY, J. (1961) A toxic factor in Brazilian groundnut meal. Veterinary Record 73, 428-429
279
BANCROFT, J. B. & STEVENS, A. (1982) Theory and Practice of Histological Techniques. New York, Churchill Livingstone. p223 BOE 246 (23562) de fecha 14.X. Anejo 7. (1981) M6todos oficiales de an~ilisis de piensos y sus primeras materias y toma de muestras BRYDEN, W. L. & CUMMING, R. B. (1980) Observations on the liver of the chicken followingaflatoxin Bl ingestion.Avian Pathology 9, 539-550 CHEN, C., PEARSON, A. M., COLEMAN, T. H., GRAY, J. I. & WOLZAK, A. M. (1985) Broiler aflatoxiensis with recovery after replacement of the contaminated diet. British Poultry Science 26, 65-71 DALVI, R. R. (1986) An overview of aflatoxicosis of poultry: Its characteristics, prevention and reduction. Veterinary Research CommunicatiOns 10, 429--443 DONALDSON, W. E., TUNG, H.-T. & HAMILTON, P. B. (1972) Depression of fatty acid synthesis in chick fiver (Gallus domesticus) by aflatoxin. Compendium of Biochemical Physiology 41B, 843-847 GIAMBRONE, J. J., DIENER, U. L., DAVIS, N. D., PANANGALA,V. S. & HOERR, F. J. (1985) Effects of aflatoxin on young turkeys and broiler chickens. Poultry Science 64, 1678-1684 GIMENO, A. (1988) Dctoxificaci6n fisiol6gica de la aflatoxina B l utilizando aditivos adsorbentes en el pienso. XXVI Symposium de Avicultura Reus. The World's Poultry ScienceAssociation. Secci6n espaflola, pp167-183 GIMENO, A. & MARTINS, L. (1983) Toxicidad y control de micotoxinas. A VIFAC 21, 25-5"1 HUFF, W. E., KUBENA, L. F., HARVEY, R. B, CORRIER, D. E. & MOLLENHAUER,H. H. (1986) Progression of aflatoxicosis in broiler chickens. Poultry Science 65, 1891-1899 MERKLEY, J. W., MAXWELL, R. J., PHILLIPS, J. G. & HUFF, W. E. (1987) Hepatic fatty acid profiles in aflatoxin-exposed broiler chickens. Poultry Science 66, 59-67 SILLER, W. G. & OSTLER, D. C. (1961) The histopathology of an enterohepatic syndrome of turkey poults. Veterinary Record 73, 134-138 SINHA, R. R. P. & ARORA, S. P. (1987) Biochemical and histopathological changes in liver of chicks fed on aflatoxin containing diets after detoxification. Indian Journal of Animal Nutrition 4, 12-19 TUNG, H.-T., DONALDSON, W. E. & HAMILTON, P. B. (1972) Altered lipid transport during aflatoxicosis. Toxicology andApplied Pharmacology 22, 97-104 VENKATA, K., GOPALAKRISHNA, D. & RAMA, P. (1988) Sequential gross and histological changes of bursa and thymus in acute and chronic experimental aflatoxicosis of broiler birds. Indian Journal of Animal Science 58, 1011-1018
Received March 11, 1991 Accepted November 25, 1991
Pathological lesions following an experimental intoxication with aflatoxin B1 in broiler chickens Y. ESPADA, M. DOMINGO, J. GOMEZ, M. A. CALVO, Department of Veterinary Medicine.
Faculty of Veterinary Science, Universitat Aut6noma de Barcelona, 08193 - Bellaterra, Barcelona,
Spain
A follow-up of chickens dosed orally over 21 days with 0-2 and 3 ~tg of aflatoxin B 1 (AFB1) g-I of bodyweight daily and their subsequent recovery 10 days after withdrawal of contaminated food was conducted. Vacuolation of liver cells during the initial days of the intoxication and cellular depletion in the follicle medulla of the bursa of Fabricius were the lesions which appeared first and persisted during the recovery phase in both groups of intoxicated animals. The intensity of these lesions and their persistence was related to the dose of aflatoxin ingested. A significant reduction in the bodyweight and absolute weights of fiver, bursa of Fabricius spleen and thyroid was observed in the higher dose group. IN 1961 Allcroft et al identified aflatoxins as the causal agent of the death of 100,000 poultry in Great Britain. These are secondary toxic metabolites and were elaborated and accumulated mainly by Aspergillus flavus and Aspergillus parasiticus. The ingestion of grain contaminated with aflatoxins by animals causes an aflatoxicosis, the appearance of which depends on the animal species, strain, age, nutritional state and state of health, the quantity of aflatoxin ingested and the duration of exposure to the toxin, as well as the presence of concomitant diseases and other mycotoxins (Gimeno and Martins 1983). The clinical condition and lesions, which have been widely described, are characterised by a reduction in bodyweight (Siller and Ostler 1961), deaths with nervous seizures and opisthotonos (Dalvi 1986), vacuolation of hepatic cells (Chen et al 1985, Merkley et al 1987), bile-duct proliferation (Siller and Ostler 1961, Gimeno 1988) and periportal fibrosis (Sinha and Arora 1987).
However, the chronological study of the process may be useful to establish the time sequence of appearance of lesions, as well as the persistence of some lesions and not others, after removing the toxin, from the point of view of diagnostic interest. Materials and methods
Aflatoxin and chicks Pure aflatoxin B t(AFBl), obtained from Sigma (St Louis), was dissolved in a small volume of a 25 per cent ethanol solution and the final dose was prepared with a 0.75 per cent carboxymethyl cellulose solution. The aflatoxin was administered orally. Day-old broiler chicks (150) weighing 40 to 45 g were obtained from a local hatchery. They were divided into three groups and each bird weighed daily: group C contained 50 chicks and served as the control, group D02 containing 50 chicks received 0.2 ~tg AFBl g-1 of bodyweight each day for 21 days and group D3 containing 50 chicks received 3 ~tg AFB1 g-1 of bodyweight each day, also for 21 days. After 21 days all birds were kept 10 more days without receiving further toxin. The starter and grower feed for the experimental birds were tested for any possible residual aflatoxin, following the official method of sampling and analysis of feed and raw materials according to Spanish legislation (BOE 246, 1981). Water and feed were given ad libitum and the animals were exposed daily to 18 hours of light. From the sixth until the 30th day groups of three animals were killed by decapitation on alternate days. All animals were weighed before being
275
276
Y. Espada, M. Domingo, J. Gomez, M. A. Calvo
killed. A detailed necropsy was then conducted. The liver, spleen, bursa of Fabricius and thymus were recovered and weighed. Tissue samples from these organs and from the kidneys, lungs, cerebrum, cerebellum, proventriculus, duodenum with pancreas, myocardium and thigh muscle were collected in 10 per cent neutral buffered formalin. After fu~ation, samples were dehydrated in ethanol, cleared in xylene and embedded in paraplast at 50°C. Tissue sections 5 ~ma thick were stained with haematoxylin and eosin. Formol fixed liver blocks were sectioned by cryostat and stained for lipids using oil red O following the technique of Bancroft and Stevens (1977). Results
Macroscopic and microscopic lesions In birds of group D3, macroscopic lesions of the liver consisted of pale, yellowish discoloration of the whole organ appearing at the sixth day of intoxication and disappearing at the eighth day after withdrawal. There was enlargement of the gallbladder (from the sixth to the 14th day of intoxication), and a fine irregular surface (from the 20th day of intoxication to the sixth day after withdrawal). Microscopically, in this group, hepatic cell vacuolation with fatty infiltration was demonstrated with oil red O, surrounding the
centrilobular hepatic veins from the sixth day of intoxication. Fatty infiltration progressively extended to the whole hepatic parenchyma (by the 12th day of intoxication), and persisted nine to 10 days after withdrawal. Concomitantly with the fatty infiltration, more basophilic cells were arranged in acini around the portal areas (Fig 1). Both lesions were found in the same region and a gradual change was observed in the staining. Mild fibrosis around portal areas was only seen in some chickens on the 18th and 20th days of intoxication. Disappearance of acini ofbasophilic cells was observed on the second day after withdrawal, whereas the fatty infiltration persisted, in decreasing amount, until the end of the experiment (10 days after withdrawal). In group D02, a pale discoloration of the liver from the 16th day of intoxication to the sixth day of withdrawal was the only macroscopic lesion observed. Microscopically, fatty infiltration of liver cells became evident on the 14th day of intoxication, disappearing on the sixth day after withdrawal. In the bursa of Fabricius of group D3 a reduction in the number of cells present in the centre of the follicle was observed from the sixth day of investigation and persisted during the recovery period. Pyknosis and karyorrhexis of lymphoid cells were occasionally seen (Fig 2). A less frequent lesion of the bursa was the formation of cysts of
FIG 1 : Liver of chicken fed with 3 ~g aflatoxin B 1 g-1 bodyweight, 21 st day of intoxication. Note formation of bile ducts around a portal vessel. × 150
Aflatoxicosis in chickens
277
FIG 2: Bursa of Fabricius of chicken fed with 3 ~g aflatoxin B 1 g-1 bodyweight, 12th day of intoxication. The medullary zone of the follicles is depleted and shows pyknosis and karyorrhexis of lymphoid cells, × 150
cylindrical and cuboidal epithelium of varying sizes, located nearby or attached to the inner epithelium lining of the bursa. In group D02 cellular depletion of the bursa of Fabricius appeared on the eighth day of intoxication with recovery on the fifth day. The other lesions described in group D3 appeared only occasionally. In the kidney on the 12th day of intoxication there was evidence of an irregular dilation of some proximal tubules without evidence of cell degeneration; the lesion regressed rapidly on removal of toxin (four days). This lesion was observed only in group D3. Other microscopic lesions occasionally observed in group D3 during the intoxication period were a slight depletion of lymphoid cells in the spleen, and hyperaemia and focal haemorrhage in the medulla of the thymus. In the rest of the samples studied no lesions were observed that could be interpreted as characteristic of the process.
Absolute weight of the organs The absolute weights of the liver, spleen, thymus and bursa of Fabricius in the three groups are shown in Fig 3. A significant reduction of their weight gain throughout the intoxication period and weight gain during the recovery phase
was observed in chickens of group D3. The mean bodyweights are shown in Fig 4.
Mortality During the intoxication period 11 chicks from the D3 group died and were not included in the results because of post mortem changes. However, the same number were omitted from the other two groups at the end of the experiment. Discussion
The hepatic lesions of aflatoxicosis described as vacuolation of hepatic cells due to fatty metamorphosis are in agreement with those reported by Chen et al (1985) and Merkley et al (1987), who classified it as degenerative changes of the liver. Bile duct proliferation considered as regenerative changes of the liver was described by Siller and Ostler (1961), Bryden and Cumming (1980) and by Gimeno (1988). In this study a greater persistence of the former was observed when the toxic material was removed, although intensity was similar for both. Cell depletion in the medulla of the follicles of the bursa of Fabricius is also a characteristic lesion. Venkata et al (1988) describe this depletion of lymphocytes in chronic aflatoxicosis (12
278
Y. Espada, M. Domingo, J. Gomez, M. A. Calvo 2424 2O
Burs
Liver 2O
15
15
~z 10. 10 5.
5
2
2
0
20
10
30
-0
10
2.80 Spleen
,~
1.80 !
20
50
Days
Days Thymus
/
2.40 2.00
0-60 1.60 1.20
~o~ 0"40
0.80 0.20 0.40 0 10
20
30
Days
0
10
20
FIG 3: Absolute weights of liver, bursa of Fabricius, spleen and thymus. • = D3 (3 ,LtgAFB1) , + --- D02 (0.2 ~tg AFB1) , Aflatoxin was administered from day 0 to 21 and the weights are the mean for three birds on each occasion
weeks), although they consider that it may vary from an intense lymphocytosis to a moderate depletion of lymphoid cells; and characterise the formation of cysts in the follicles as regressive changes of the bursa. On the other hand, Giambrone et al (1985) did not observe any influence on the development of the bursa although they considered that the immune system is a very sensitive indicator of aflatoxicosis in poultry. According to Huff et al (1986), Merkley et al (1987) and Gimeno (1988), the gain of whole bodyweight and the absolute gain in weight of the organs investigated was significantly reduced in a dose-dependent relationship. Noticeably, the rate of bodyweight gain did not recover after the withdrawal of intoxication. Aflatoxin B l induced an accumulation of lipids in the liver which caused an increase in its relative weight (data not shown) and the vacuolation of
30
Days [] =
C Control group.
hepatocytes. This has also been reported by Chen et al (1985) and Merkley et al (1987), and can be explained according to Huff et al (1986) by an increase in the concentration of liver lipids, which has been demonstrated with oil red O staining. This increase may have two origins; according to Tung et al (1972) it stems from a general inhibition of lipid transport, and according to Donaldson et al (1972) from an interference in lipogenesis. The atrophy of the bursa of Fabricius, described by Merkley et al (1987) and of the thymus, reported by Gimeno (1988) and Venkata et al (1988), confirm that the immune system is a reliable indicator of aflatoxicosis. Observations on the time sequence and intensity of the lesions in relation to the dose of aflatoxin administered show that the vacuolation of hepatocytes by fatty metamorphosis and the
Aflatoxicosis in chickens 800 t Mean bodyweight
i JC:
600
400 O t,n
2O0
0 0
10
20
30
Days FIG 4: Mean bodyweight during the investigation. • = D3 (3 ~tg AFB1), + = D02 (0-2 Ixg AFSl), [ ] = C Control group. Aflatoxin was administered from day 0 to 21. The weights are the mean for three birds on each occasion
cellular depletion in the medulla of the follicles of the bursa of Fabricius were the earliest lesions and also the most persistent lesions when the toxic source was removed. It may be concluded, therefore, that these changes could be used as indicators in those cases of suspected aflatoxicosis in which the analysis of the feed ingested cannot lead to a diagnosis as it no longer contains aflatoxins. Acknowledgement This work was supported by a grant of the fruiT, Generalitat de Catalunya. References ALLCROFT, R., CARNAGHAN, R. B., SARGEANT, K. & O'KELLY, J. (1961) A toxic factor in Brazilian groundnut meal. Veterinary Record 73, 428-429
279
BANCROFT, J. B. & STEVENS, A. (1982) Theory and Practice of Histological Techniques. New York, Churchill Livingstone. p223 BOE 246 (23562) de fecha 14.X. Anejo 7. (1981) M6todos oficiales de an~ilisis de piensos y sus primeras materias y toma de muestras BRYDEN, W. L. & CUMMING, R. B. (1980) Observations on the liver of the chicken followingaflatoxin Bl ingestion.Avian Pathology 9, 539-550 CHEN, C., PEARSON, A. M., COLEMAN, T. H., GRAY, J. I. & WOLZAK, A. M. (1985) Broiler aflatoxiensis with recovery after replacement of the contaminated diet. British Poultry Science 26, 65-71 DALVI, R. R. (1986) An overview of aflatoxicosis of poultry: Its characteristics, prevention and reduction. Veterinary Research CommunicatiOns 10, 429--443 DONALDSON, W. E., TUNG, H.-T. & HAMILTON, P. B. (1972) Depression of fatty acid synthesis in chick fiver (Gallus domesticus) by aflatoxin. Compendium of Biochemical Physiology 41B, 843-847 GIAMBRONE, J. J., DIENER, U. L., DAVIS, N. D., PANANGALA,V. S. & HOERR, F. J. (1985) Effects of aflatoxin on young turkeys and broiler chickens. Poultry Science 64, 1678-1684 GIMENO, A. (1988) Dctoxificaci6n fisiol6gica de la aflatoxina B l utilizando aditivos adsorbentes en el pienso. XXVI Symposium de Avicultura Reus. The World's Poultry ScienceAssociation. Secci6n espaflola, pp167-183 GIMENO, A. & MARTINS, L. (1983) Toxicidad y control de micotoxinas. A VIFAC 21, 25-5"1 HUFF, W. E., KUBENA, L. F., HARVEY, R. B, CORRIER, D. E. & MOLLENHAUER,H. H. (1986) Progression of aflatoxicosis in broiler chickens. Poultry Science 65, 1891-1899 MERKLEY, J. W., MAXWELL, R. J., PHILLIPS, J. G. & HUFF, W. E. (1987) Hepatic fatty acid profiles in aflatoxin-exposed broiler chickens. Poultry Science 66, 59-67 SILLER, W. G. & OSTLER, D. C. (1961) The histopathology of an enterohepatic syndrome of turkey poults. Veterinary Record 73, 134-138 SINHA, R. R. P. & ARORA, S. P. (1987) Biochemical and histopathological changes in liver of chicks fed on aflatoxin containing diets after detoxification. Indian Journal of Animal Nutrition 4, 12-19 TUNG, H.-T., DONALDSON, W. E. & HAMILTON, P. B. (1972) Altered lipid transport during aflatoxicosis. Toxicology andApplied Pharmacology 22, 97-104 VENKATA, K., GOPALAKRISHNA, D. & RAMA, P. (1988) Sequential gross and histological changes of bursa and thymus in acute and chronic experimental aflatoxicosis of broiler birds. Indian Journal of Animal Science 58, 1011-1018
Received March 11, 1991 Accepted November 25, 1991
