317
REGULATION OF P INTAKE
Mostert, G. C , and L. G. Swart, 1968. Calcium and phosphorus levels in laying rations of hens. S. Afr. J. Agr. Sci. 11:687-697. O'Rourke, W. F., H. R. Bird, P. H. Phillips and W. W. Cravens, 1954. The effects of low phosphorus rations on egg production and hatchability. Poultry Sci. 33: 1117-1122. Roland, D. A., Sr., D. R. Sloan and R. H. Harms, 1971. An easy and inexpensive system for measuring individual feed consumption. Poultry Sci. 50: 12271228. Salvesen, H. A., A. B. Hastings and J. F. Mcintosh, 1924. Blood changes and clinical symptoms following oral administration of phosphates. J. Biol. Chem. 60: 311-326. Taylor, T. G., 1965. Dietary phosphorus and eggshell thickness in the domestic fowl. Br. Poultry Sci. 6: 79-87. Walter, E. D., and J. R. Aitken, 1962. Phosphorus requirements of laying hens confined to cages. Poultry Sci. 41: 386-390.
Ultrastructural Changes Induced by Chronic Aflatoxicosis in Chickens V. C.
K E L L E Y 1 AND E . C.
MORA
Department of Poultry Science, Auburn University Agricultural Experiment Station, Auburn, 36830
Alabama,
(Received for publication May 16, 1975)
ABSTRACT Effects in chickens of a low concentration of aflatoxin in the diet were investigated. After eight weeks, heart, pancreas, and liver tissues were examined by electron microscopy. Most damage was to liver; changes were observed both in individual parenchymal cells and in tissue organization. Proliferation of bile duct epithelial cells was extensive. The pancreas showed little change and was, therefore, a good organ for observation of early and/or mild ultrastructural changes associated with aflatoxicosis. The priority of initial nuclear change over cytoplasmic change was indicated in this organ. In the myocardium, the only significant changes were in mitochondria. Mitochondria of liver and pancreas were not altered. Leukoviruses were observed in the tissues of one aflatoxin-treated bird. POULTRY SCIENCE 55: 317-324, 1976
INTRODUCTION FLATOXINS are fungal metabolites that cause specific debilitating and often fatal lesions in many animals and are the most
A
1. Present address: Dept. of Botany and Microbiology, Auburn University, Auburn, Ala. 36830.
potent, naturally occurring carcinogens known (Wogan and Newberne, 1967). In susceptible species, young animals have been shown to be more sensitive than the old and the principle site of acute toxicity is the liver (Goldblatt, 1969). Low concentrations of aflatoxin, administered over extended peri-
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on June 9, 2016
Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics, 11: 1-42. Evans, R. J., J. S. Carver and A. W. Brant, 1944. The influence of dietary factors on eggshell quality: 1. Phosphorus. Poultry Sci. 23: 9-15. Gardiner, E. E., 1973. Inorganic phosphorus, organic phosphorus and inorganic calcium in blood plasma from two breeds of chickens fed various levels of dietary calcium and phosphorus. Can. J. An. Sci. 53: 551-556. Harms, R. H., B. L. Damron and P. W. Waldroup, 1965. Influence of high phosphorus levels in caged layer diets. Poultry Sci. 44: 1249-1253. Hinners, S. W., J. T. Gholson and M. L. Ritchason, 1963. The effect of varying levels of calcium and phosphorus on egg production and egg quality. Poultry Sci. 42: 1277. Holcombe, D. J., D. A. Roland, Sr. and R. H. Harms, 1975. The ability of hens to adjust calcium intake when given a choice of diets containing two levels of calcium. Poultry Sci. 54: 552-561. Hunt, J. R., and H. W. R. Chancey, 1970. Influence of dietary phosphorus on shell quality. Br. Poultry Sci. 11:259-267.
318
V. C. KELLEY AND E. C. MORA
except for the aflatoxin content. The ration for treated birds was assayed by the method of Pons and Goldblatt (1965) and contained approximately 390 p.p.b. aflatoxin B , equivalent. Gross pathology of these birds has been reported (Rivelli-Florentin et al., 1969). Birds were sacrificed at 8 weeks of age and heart, pancreas, and liver tissue taken at random from control and treated animals. All tissues were fixed in 3% glutaraldehyde, post-fixed in osmium tetroxide, dehydrated in graded alcohols, and embedded in EponAraldite. Thin sections were stained with uranyl acetate and lead citrate and examined with a Philips 300 electron microscope. RESULTS No consistent differences in aflatoxin susceptibility among the three breeds were observed. The results will therefore be concerned with these birds as a single group. A. Heart. Myocardial tissue of the control group was characterized by distinctly banded myofibrils interspersed with numerous large mitochondria (Fig. 1). Within the mitochondria there were regions of a homogenous granular electron dense matrix material as
•V
MATERIALS AND METHODS Day-old chickens of the Langshan breed, Delaware x New Hampshire crossbreed, and Cornish x White Plymouth Rock crossbreed were divided into treatment and control groups. Feed for the six groups was identical
FIG. 1. Heart specimen from untreated bird. The linear sarcomere units (S) are joined to form myofibrils. Mitochondria (M) are present between myofibrils. Glycogen granules (gl) are present between other organelles, x 29,500
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on June 9, 2016
ods of time or as single doses have resulted in the development of hepatomas in several species (Barnes, 1967; Carnaghan, 1967). Aflatoxin toxicity has been demonstrated in many farm animals including cattle, swine, and poultry; turkey poults and ducklings are quite susceptible, but chickens have been considered to be relatively resistant (Goldblatt, 1969). However, chickens have been shown to be affected by aflatoxin, both in naturally occurring field poisonings and in controlled feeding studies. Aflatoxicosis in chickens has resulted in marked decrease in egg production (Hamilton, 1971), growth retardation and hepatic damage (Hamilton, 1971; Kratzer et al., 1969; Pier et al., 1970); decreased immunity to fowl chloera (Pier et al., 1970) and increased susceptibility to Marek's disease and coccidiosis (Edds et al., 1973). Significant mortality in chickens has also been caused by aflatoxin (Hamilton, 1971). The biochemical mechanism by which aflatoxin causes these toxic reactions or induces carcinomas in certain animals has never been satisfactorily explained. A growing body of evidence suggests that aflatoxin binds to DNA and then inhibits RNA polymerase action (Wogan, 1974). Ultrastructural studies often have been valuable in the interpretation of such biochemical studies. With aflatoxin-treated chickens, electron microscope investigations have not been carried out, though light microscopic studies have been made. The purpose of this electron microscope study was to determine any alterations in tissues of chickens fed a low level of dietary aflatoxin.
CHRONIC AFLATOXICOSIS
319
FIG. 3. Heart specimen from aflatoxin-treated group. Mitochondrion containing a myelinlike figure (my). A series of fine parallel electron dense filaments and a vacuole are present, x 53,900.
2
500 nm
FIG. 2. Heart specimen from aflatoxin-treated group. Mitochondrion containing neutral lipid-like inclusions. Cristae (c) are still visible in some areas of mitochondrion, while they are replaced by the lipid-like bodies (L) in other regions, x 42,200.
with inclusions (Fig. 3). Apart from the lipidlike inclusions in the mitochondria, there was no detectable increase in heart tissue lipid. Two other changes were observed in the hearts from treated birds, each occurring in a single specimen. In one specimen there was evidence of intracardial edema. In another specimen, virus particles were found in myocardium, liver, and pancreas (Fig. 4). These virions were spherical to ovoid and approximately 100-120 nm. in diameter. The particles had an electron-dense nucleoid surrounded by material of lower density, and were bound by a narrow membrane covered with spikes. In heart, the virions were situated in spaces
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on June 9, 2016
well as regions of well-defined cristae. Lipid droplets were frequently observed in close proximity to the mitochondria. Inaflatoxin-fed birds, several changes were observable in the mitochondria. Most striking was the presence of numerous intramitochondrial inclusions. The inclusions were of two types, both of which appeared to be lipid in nature. The first type was spherical to oval and homogeneously electron dense (Fig. 2); the second type was a series of parallel filaments (Fig. 3). Frequently, these oval and myelin-like inclusions were found in the same mitochondrion. Occasionally, vacuoles were observed in mitochondria but only in those
320
V. C. KELLEY AND E. C. MORA
" y x/y #•
men was somewhat dilated. Other specimens did not show similar prominent or extensive dilations. There was no obvious difference in zymogen granule production in the two groups. There were no differences between mitochondria of the control and treated groups, and lipid inclusions were not observed.
100""'
few
.
...
y:M''-"-*'•"••
around the myofibrils and mitochondria and were never observed within sarcomeres or mitochondria. In liver and pancreas, the virions were always extracellular. The tissue surrounding these particles was not visibly altered. B. Pancreas. Exocrine pancreas was chosen for this study. The most consistent change was in the nucleoli of the aflatoxin-fed group. In acinar nuclei of the treated animals, nucleoli were frequently divided into granular and fibrillar components (Fig. 5). The rough endoplasmic reticulum (ER) of one speci-
FIG. 5. Acinar cell from aflatoxin-treated pancreas. Segregation of nucleolus into granular (gr) and fibrillar (fb) components, x 22,900.
••'?'•'
-
'
N
- ' : ' .
•^
V
;
i . ,
" ;
-
"
>
. 4*. >-
•
•'''•
-' •
'•$
M
FIG. 6. Normal liver parenchymal cell from untreated group. Prominent in this figure are the nucleus (N), nucleolus (Nuc), mitochondria (M) and glycogen masses (gl). x 10,700.
FIG. 7. Aflatoxin treated hepatic cell with dilated cisternae (c). The vacuolar distensions in the cytoplasm are oval and relatively uniform in size, x 19,600.
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on June 9, 2016
FIG. 4. Pancreas specimen from aflatoxin group. High magnification of virus particles. The dense central nucleoid is surrounded by a material of lower density and surface projections are present on the outer membrane, x 108.800.
C. Liver. In the aflatoxin-treated group. changes were detected both in individual cells and in tissue organization. While cisternae
321
CHRONIC AFLATOXICOSIS
FIG. 8. Liver from aflatoxin-treated bird showing numerous large vacuoles (Vac), x 12,200.
FIG. 9. Liver tissue of aflatoxin-treated group showing bile ductule. Three cells and part of a fourth comprise this section of the ductule, x 14,500. concomitant increase in connective tissue nuclei and collagen fibrils. Collagen bundles were found in the livers of some treated birds. Large numbers of heterophils, lymphocytes, and plasma cells were present in liver of all treated birds. DISCUSSION In heart tissue, the most outstanding ultrastructural changes were observed in mitochondria. The lipid-like mitochondrial inclusions were similar to inclusions described in human liver mitochondria by Rouiller and Jezequel (1963), who concluded that the droplets were lipid. The intramitochondrial vacuoles observed (Fig. 3) were similar to the membrane-limited matrix cavities of mitochondria found in rat liver 24 hours following aflatoxin administration (Svoboda et al., 1966). The presence of lipid inclusions in mitochondria could reflect general disturbance in lipid metabolism, but this would appear unlikely since the inclusions were not found in pancreas and liver. This is possibly related to the intense respiratory activity of heart mitochondria. The virions observed in the heart of one aflatoxin-treated bird were also found in the pancreas and liver of the same bird (Fig. 4). The virus particles resemble the leukosis
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on June 9, 2016
of the ER in the control group were not visibly dilated (Fig. 6), all of the treated group tissue had some degree of cisternal dilation (Fig. 7). There were a few ribosomes adherent to the cisternal membranes but there was no continuous ribosomal lining such as is found for control ER. Parenchymal hepatic cells of the aflatoxintreated group had large vacuoles which were not membrane-bound and of extreme size variation (Fig. 8). Some of the moderate sized vacuoles appeared to coalesce to form large vacuoles. Intranuclear change was minimal. Nucleolar segregation was occasionally seen in aflatoxin-treated livers, but not as frequently as in the pancreas. The most striking changes in aflatoxintreated liver were in tissue organization. Bile duct proliferation, the most common lesion in acute aflatoxicosis, was confirmed for chickens in this study. In some areas of the treated tissues, proliferating ductules were surrounded by connective tissue and cells of inflammation. Usually the ductules were composed of three or more cells joined to form a lumen (Fig. 9). In areas where normal tissue organization had been lost, bile duct epithelial cells could be seen alone or in pairs forming incomplete ductules. There was a vast increase in inflammatory cells with a
322
V. C. KELLEY AND E. C. MORA
of Pong and Wogan (1970), who found that nucleolar segregation and RNA polymerase inhibition, observed up to 12 hr. following administration of aflatoxin, had disappeared within 36 hr. In the present study, segregated nucleoli were observed much less frequently in liver than in pancreas. Since this was a chronic feeding study, the presence of segregated nucleoli would not be expected. Their presence in pancreas, which is otherwise changed little, suggests that pancreas may exhibit some of the basic liver alterations but on a much slower timetable. It might then prove to be a good model tissue to determine the sequence of ultrastructural changes. Like nucleolar segregation, dilation of the ER cisternae is an early change of aflatoxicosis. In the aflatoxin-treated chickens of this study, cisternal dilations were observed in the ER of liver and pancreas, with the hepatic dilations being larger, more numerous, and more homogeneous (Fig. 7). While degranulation of rough ER was not noticeable in the pancreas, cisternae of liver cells were membrane bound and few if any ribosomes were attached. Butler (1966) found cisternal dilation to be the earliest change in rat liver. Similar results were obtained by Svoboda et al. (1966) with rats and by Theron (1965) with ducklings. Increased prominence of the smooth ER was a change consistently observed in the liver of rats in a chronic feeding study (Svoboda et al., 1966). Otherwise dilation of cisternae of ER has been associated with acute cytoplasmic changes. The cytoplasmic changes related to RNA and protein synthesis are believed to occur following a primary nuclear alteration (Svoboda and Higginson, 1968). This was consistent with the lesser degree of cytoplasmic change observed in pancreas, which was still in a stage of primarily nuclear change. In accord with the general finding that liver is the primary site of damage in aflatoxin poisoning of several species, the most exten-
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on June 9, 2016
viruses, a group of RNA viruses originally discovered in chickens as a cause of leukemias and solid tumors. There are at least three possible explanations for the presence of these viruses in the aflatoxin-treated chicken. First, their occurence in an aflatoxin-treated bird may be coincidental, since these leukoviruses have been found in apparently healthy chickens (Temin, 1972). Second, the viruses may have reached detectable levels within treated birds due to a decrease in immunity to microorganisms; this would correlate with a reported decrease in immunity to fowl cholera (Pier et al., 1970), Marek's disease, and coccidiosis (Edds et al., 1973) attributable to aflatoxicosis. A third possibility is induction of a latent avian leukovirus by aflatoxin. Such a phenomenon is not unknown, since chemical carcinogens have been shown to induce avian leukoviruses in normal chicken cells (Weiss et al., 1971). In the present study, there were no macroscopic changes in pancreas (Rivelli-Florentin, 1969) and pancreatic damage reported by others working with aflatoxin has not been severe (Asplin and Carnaghan, 1961; Diener et al., 1963). The most prominent change in pancreatic acinar cells of aflatoxin-treated chickens was nuclear segregation or "capping" (Fig. 5), a phenomenon shown by time-lapse photography to be due to a redistribution of the morphological components of the nucleolus (Reynolds et al., 1963). Nucleolar segregation into granular and fibrillar components has been a frequent change induced in liver by aflatoxin and several other chemical carcinogens (Svoboda and Higginson, 1968); it has been related to the inhibition of DNA-dependent RNA-polymerase (Pong and Wogan, 1970). The separation was found to be one of the immediate ultrastructural changes following administration of aflatoxin, but was not associated in studies of chronic aflatoxicosis in liver (Butler, 1966; Svoboda et al, 1966). Those observations are in agreement with the studies
323
CHRONIC AFLATOXICOSIS
but not in the later stages. This suggested that destruction was less advanced in those specimens with the complete ductules. In many areas of the aflatoxin-treated livers, parenchymal cells had been almost completely replaced by invading mononuclear and polymorphonuclear cells. This was similar to the conditions described by Carnaghan et al. (1966), in which islands of lymphocytic hyperplasia surrounded areas of degenerating duct cells. Normal parenchymal cells could be found in a few areas but these usually were not invaded by inflammatory cells, presenting the possibility that these might be areas of regenerating parenchymal cells. The small bundles of collagen observed in liver confirm previous observations (Goldblatt, 1969) of a mild to moderate fibrosis in turkeys, chickens, ducklings, and other animals. Extravascular erythrocytes were found only in areas of extensive tissue damage. In no case was there evidence of ultrastructural abnormalities in organelles that were in contact with erythrocytes. This was in contrast to the report of Theron (1965), who found, in acute toxicity studies, that organelles in contact with extravascular erythrocytes were specifically altered. Other workers (Svoboda et al., 1966; Butler, 1966) have not observed this phenomenon. REFERENCES Asplin, F. D., and R. B. A. Carnaghan, 1961. The toxicity of certain groundnut meals for poultry with special reference to their effect on ducklings and chickens. Vet. Rec. 73: 1215-1218. Barnes, J. M., 1967. Toxic fungi with special reference to aflatoxin. Trop. Sci. 9: 64-74. Butler, W. H., 1964. Acute liver injury in ducklings as a result of aflatoxin poisoning. J. Path. Bacteriol. 88: 189-196. Butler, W. H., 1966. Early hepatic parenchymal changes induced in the rat by aflatoxin B,. Amer. J. Pathol. 49: 113-128. Carnaghan, R. B. A., 1967. Hepatic tumors and other
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on June 9, 2016
sive pathological changes observed in this study were in the liver. Livers from chickens on an aflatoxin diet all had some degree of vacuolation of the parenchymal cells (Fig. 8). Vacuolation of liver parenchymal cells has been found to be associated with aflatoxicosis of turkeys (Wannop, 1961), ducklings (Madhaven and Rao, 1966), and chickens (Carnaghan et al., 1966). In this study, no mitochondrial abnormalities of liver and pancreas were seen. However, in the hearts of the same animals, there were dramatic mitochondrial aberrations. Early mitochondrial changes in hepatocytes have been reported in aflatoxin poisoning of rats and ducklings (Butler, 1966; Theron, 1965) but have not been a feature of chronic toxicity studies. These data suggest that mitochondria are affected only secondarily and can recover. Possibly, toxicity to pancreas was too slight to be reflected in mitochondrial abnormalities. The pattern of bile duct epithelial proliferation in chicken was similar to that in duckling (Butler, 1964) and swine (Loosmore and Harding, 1961). Studies of Rhode Island Red chicks fed rations containing 1500 p.p.b. of aflatoxin B , showed slight proliferation of bile duct epithelial cells within 3-1/2 days (Carnaghan et al., 1966). Within 2 weeks, bile duct proliferation was prominent and increased through the sixth week. Because the level of aflatoxin used by Carnaghan was nearly four times that used in this study, the degree of proliferation of bile duct epithelium reported here after 8 weeks possibly does not represent the maximal possible proliferation. In only one treated chicken were complete ductules numerous (Fig. 9). In all treated groups, single cells tentatively identified as bile duct epithelium or, less frequently, pairs of ductular cells forming incomplete ductules were numerous in damaged areas. Butler (1964) observed the formation of complete ductules in the early stages of aflatoxicosis
324
V. C. K E L L E Y AND E . C.
Reynolds, R. C , P. O.' B. Montgomery and D. H. Karney, 1963. Nucleolar "caps"-a morphologic entity produced by the carcinogen 4-nitroquinoline N-oxide. Cancer Res. 23: 535-538. Rivelli-Florentin, E., 1969. The effects of aflatoxin on different breeds and strains of chickens. M.S. Thesis, Auburn Univ., Auburn, Ala. Rivelli-Florentin, E., G. J. Cottier, U. L. Diener and N. D. Davis. 1969. The effect of aflatoxin on different breeds and crosses of chickens. Poultry Sci. 48: 1809. Rouiller, Ch., and A. M. Jezequil, 1963. Electron microscopy of the liver, p. 195-264. In: The Liver, Ch. Rouiller (ed.), vol. 1. Academic Press, New York. Svoboda, D. J., H. J. Grady and J. Higginson, 1966. Aflatoxin B, injury in rat and monkey liver. Amer. J. Pathol. 49: 1023-1051. Svoboda, D., and J. Higginson, 1968. A comparison of ultrastructural changes in rat liver due to chemical carcinogens. Cancer Res. 28: 1703-1733. Theron, J. J., 1965. Acute liver injury in ducklings as a result of aflatoxin poisoning. Lab. Invest. 14: 1586-1603. Temin, H., 1972. The RNA tumor viruses. Background and foreground. Proc. Nat. Acad. Sci. 69: 10161020. Wannop, C. C , 1961. The histopathology of turkey " X " disease in Great Britain. Avian Dis. 5: 371-381. Weiss, R. A., R. R. Friis, R. Katz and P. K. Vogt, 1971. Induction of avian tumor viruses in normal cells by physical and chemical carcinogens. Virology, 46: 920-938. Wogan, G. N., 1974. Biochemical effects of aflatoxins. Israel J. Med. Sci. 10: 441-445. Wogan, G. N., and P. M. Newberne, 1967. Dose-response characteristics of Aflatoxin B carcinogenesis in the rat. Cancer Res. 27: 2370-2376.
NEWS AND NOTES (Continued from page 295) Huck has assumed overall direction for the Company's pharmaceutical and animal health divisions in the United States and Canada, its industrial and environmental health businesses and its consumer products business. Thomas B. Davis has been elected Vice President and General Manager of the Division that retains the Merck Chemical Division designation. It was formerly
the industrial and fine chemicals area. Spencer A. Stouffer has been elected Vice President and General Manager of Merck Chemical Manufacturing Division, formerly the operations area. ELANCO NOTES Robert E. Howerton has been promoted to Executive Director, Marketing-Animal Products for Elanco
(Continued on page 340)
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on June 9, 2016
chronic liver changes in rats following a single oral administration of aflatoxin. Brit. J. Cancer, 28: 811-814. Carnaghan, R. B. A., G. Lewis, D. S. P. Patterson and R. Allcroft, 1966. Biochemical and pathological aspects of groundnut poisoning in chickens. Pathol. Vet. 3: 601-615. Diener, U. L., N. D. Davis, W. D. Salmon and C. O. Prickett, 1963. Toxin-producing Aspergillis isolated from domestic peanuts. Science, 142: 14911492. Edds, G. T., K. P. C. Nair and C. F. Simpson, 1973. Effect of aflatoxin B, on resistance in poultry against cecal coccidiosis and Marek's disease. Amer. J. Vet. Res. 34: 819-826. Goldblatt, L. A., 1969. Aflatoxin. Academic Press, New York. Hamilton, P. B., 1971. A natural and severe occurrence of aflatoxicosis in laying hens. Poultry Sci. 50: 1880-1882. Kratzer, F. H., D. Bandy, M. Wiley and A. N. Booth. 1969. Aflatoxin effects in poultry. Proc. Soc. Exptl. Biol. Med. 131: 1281-1284. Loosmore, R. M., and J. D. J. Harding, 1961. A toxic factor in Brazilian groundnut meal causing liver damage in pigs. Vet. Rec. 73: 1362-1364. Madhaven.T. V., andK. Rao, 1966. Hepatic infarction in ducklings in aflatoxin poisoning. Arch. Pathol. 81:520-524. Pier, A. C , and K. L. Heddleston, 1970. The effect of aflatoxin on immunity in turkeys to bacterial challenge. Avian Dis. 14: 797-809. Pong, R. S., and G. N. Wogan, 1970. Time course and dose-response characteristics of aflatoxin B, effects on rat liver RNA polymerase and ultrastructure. Cancer Res. 30: 294-304. Pons, W. A., and L. A. Goldblatt, 1965. The determination of aflatoxins in cottonseed products. J. Amer. Oil. Chem. Soc. 42: 471-475.
MORA
REGULATION OF P INTAKE
Mostert, G. C , and L. G. Swart, 1968. Calcium and phosphorus levels in laying rations of hens. S. Afr. J. Agr. Sci. 11:687-697. O'Rourke, W. F., H. R. Bird, P. H. Phillips and W. W. Cravens, 1954. The effects of low phosphorus rations on egg production and hatchability. Poultry Sci. 33: 1117-1122. Roland, D. A., Sr., D. R. Sloan and R. H. Harms, 1971. An easy and inexpensive system for measuring individual feed consumption. Poultry Sci. 50: 12271228. Salvesen, H. A., A. B. Hastings and J. F. Mcintosh, 1924. Blood changes and clinical symptoms following oral administration of phosphates. J. Biol. Chem. 60: 311-326. Taylor, T. G., 1965. Dietary phosphorus and eggshell thickness in the domestic fowl. Br. Poultry Sci. 6: 79-87. Walter, E. D., and J. R. Aitken, 1962. Phosphorus requirements of laying hens confined to cages. Poultry Sci. 41: 386-390.
Ultrastructural Changes Induced by Chronic Aflatoxicosis in Chickens V. C.
K E L L E Y 1 AND E . C.
MORA
Department of Poultry Science, Auburn University Agricultural Experiment Station, Auburn, 36830
Alabama,
(Received for publication May 16, 1975)
ABSTRACT Effects in chickens of a low concentration of aflatoxin in the diet were investigated. After eight weeks, heart, pancreas, and liver tissues were examined by electron microscopy. Most damage was to liver; changes were observed both in individual parenchymal cells and in tissue organization. Proliferation of bile duct epithelial cells was extensive. The pancreas showed little change and was, therefore, a good organ for observation of early and/or mild ultrastructural changes associated with aflatoxicosis. The priority of initial nuclear change over cytoplasmic change was indicated in this organ. In the myocardium, the only significant changes were in mitochondria. Mitochondria of liver and pancreas were not altered. Leukoviruses were observed in the tissues of one aflatoxin-treated bird. POULTRY SCIENCE 55: 317-324, 1976
INTRODUCTION FLATOXINS are fungal metabolites that cause specific debilitating and often fatal lesions in many animals and are the most
A
1. Present address: Dept. of Botany and Microbiology, Auburn University, Auburn, Ala. 36830.
potent, naturally occurring carcinogens known (Wogan and Newberne, 1967). In susceptible species, young animals have been shown to be more sensitive than the old and the principle site of acute toxicity is the liver (Goldblatt, 1969). Low concentrations of aflatoxin, administered over extended peri-
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on June 9, 2016
Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics, 11: 1-42. Evans, R. J., J. S. Carver and A. W. Brant, 1944. The influence of dietary factors on eggshell quality: 1. Phosphorus. Poultry Sci. 23: 9-15. Gardiner, E. E., 1973. Inorganic phosphorus, organic phosphorus and inorganic calcium in blood plasma from two breeds of chickens fed various levels of dietary calcium and phosphorus. Can. J. An. Sci. 53: 551-556. Harms, R. H., B. L. Damron and P. W. Waldroup, 1965. Influence of high phosphorus levels in caged layer diets. Poultry Sci. 44: 1249-1253. Hinners, S. W., J. T. Gholson and M. L. Ritchason, 1963. The effect of varying levels of calcium and phosphorus on egg production and egg quality. Poultry Sci. 42: 1277. Holcombe, D. J., D. A. Roland, Sr. and R. H. Harms, 1975. The ability of hens to adjust calcium intake when given a choice of diets containing two levels of calcium. Poultry Sci. 54: 552-561. Hunt, J. R., and H. W. R. Chancey, 1970. Influence of dietary phosphorus on shell quality. Br. Poultry Sci. 11:259-267.
318
V. C. KELLEY AND E. C. MORA
except for the aflatoxin content. The ration for treated birds was assayed by the method of Pons and Goldblatt (1965) and contained approximately 390 p.p.b. aflatoxin B , equivalent. Gross pathology of these birds has been reported (Rivelli-Florentin et al., 1969). Birds were sacrificed at 8 weeks of age and heart, pancreas, and liver tissue taken at random from control and treated animals. All tissues were fixed in 3% glutaraldehyde, post-fixed in osmium tetroxide, dehydrated in graded alcohols, and embedded in EponAraldite. Thin sections were stained with uranyl acetate and lead citrate and examined with a Philips 300 electron microscope. RESULTS No consistent differences in aflatoxin susceptibility among the three breeds were observed. The results will therefore be concerned with these birds as a single group. A. Heart. Myocardial tissue of the control group was characterized by distinctly banded myofibrils interspersed with numerous large mitochondria (Fig. 1). Within the mitochondria there were regions of a homogenous granular electron dense matrix material as
•V
MATERIALS AND METHODS Day-old chickens of the Langshan breed, Delaware x New Hampshire crossbreed, and Cornish x White Plymouth Rock crossbreed were divided into treatment and control groups. Feed for the six groups was identical
FIG. 1. Heart specimen from untreated bird. The linear sarcomere units (S) are joined to form myofibrils. Mitochondria (M) are present between myofibrils. Glycogen granules (gl) are present between other organelles, x 29,500
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on June 9, 2016
ods of time or as single doses have resulted in the development of hepatomas in several species (Barnes, 1967; Carnaghan, 1967). Aflatoxin toxicity has been demonstrated in many farm animals including cattle, swine, and poultry; turkey poults and ducklings are quite susceptible, but chickens have been considered to be relatively resistant (Goldblatt, 1969). However, chickens have been shown to be affected by aflatoxin, both in naturally occurring field poisonings and in controlled feeding studies. Aflatoxicosis in chickens has resulted in marked decrease in egg production (Hamilton, 1971), growth retardation and hepatic damage (Hamilton, 1971; Kratzer et al., 1969; Pier et al., 1970); decreased immunity to fowl chloera (Pier et al., 1970) and increased susceptibility to Marek's disease and coccidiosis (Edds et al., 1973). Significant mortality in chickens has also been caused by aflatoxin (Hamilton, 1971). The biochemical mechanism by which aflatoxin causes these toxic reactions or induces carcinomas in certain animals has never been satisfactorily explained. A growing body of evidence suggests that aflatoxin binds to DNA and then inhibits RNA polymerase action (Wogan, 1974). Ultrastructural studies often have been valuable in the interpretation of such biochemical studies. With aflatoxin-treated chickens, electron microscope investigations have not been carried out, though light microscopic studies have been made. The purpose of this electron microscope study was to determine any alterations in tissues of chickens fed a low level of dietary aflatoxin.
CHRONIC AFLATOXICOSIS
319
FIG. 3. Heart specimen from aflatoxin-treated group. Mitochondrion containing a myelinlike figure (my). A series of fine parallel electron dense filaments and a vacuole are present, x 53,900.
2
500 nm
FIG. 2. Heart specimen from aflatoxin-treated group. Mitochondrion containing neutral lipid-like inclusions. Cristae (c) are still visible in some areas of mitochondrion, while they are replaced by the lipid-like bodies (L) in other regions, x 42,200.
with inclusions (Fig. 3). Apart from the lipidlike inclusions in the mitochondria, there was no detectable increase in heart tissue lipid. Two other changes were observed in the hearts from treated birds, each occurring in a single specimen. In one specimen there was evidence of intracardial edema. In another specimen, virus particles were found in myocardium, liver, and pancreas (Fig. 4). These virions were spherical to ovoid and approximately 100-120 nm. in diameter. The particles had an electron-dense nucleoid surrounded by material of lower density, and were bound by a narrow membrane covered with spikes. In heart, the virions were situated in spaces
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on June 9, 2016
well as regions of well-defined cristae. Lipid droplets were frequently observed in close proximity to the mitochondria. Inaflatoxin-fed birds, several changes were observable in the mitochondria. Most striking was the presence of numerous intramitochondrial inclusions. The inclusions were of two types, both of which appeared to be lipid in nature. The first type was spherical to oval and homogeneously electron dense (Fig. 2); the second type was a series of parallel filaments (Fig. 3). Frequently, these oval and myelin-like inclusions were found in the same mitochondrion. Occasionally, vacuoles were observed in mitochondria but only in those
320
V. C. KELLEY AND E. C. MORA
" y x/y #•
men was somewhat dilated. Other specimens did not show similar prominent or extensive dilations. There was no obvious difference in zymogen granule production in the two groups. There were no differences between mitochondria of the control and treated groups, and lipid inclusions were not observed.
100""'
few
.
...
y:M''-"-*'•"••
around the myofibrils and mitochondria and were never observed within sarcomeres or mitochondria. In liver and pancreas, the virions were always extracellular. The tissue surrounding these particles was not visibly altered. B. Pancreas. Exocrine pancreas was chosen for this study. The most consistent change was in the nucleoli of the aflatoxin-fed group. In acinar nuclei of the treated animals, nucleoli were frequently divided into granular and fibrillar components (Fig. 5). The rough endoplasmic reticulum (ER) of one speci-
FIG. 5. Acinar cell from aflatoxin-treated pancreas. Segregation of nucleolus into granular (gr) and fibrillar (fb) components, x 22,900.
••'?'•'
-
'
N
- ' : ' .
•^
V
;
i . ,
" ;
-
"
>
. 4*. >-
•
•'''•
-' •
'•$
M
FIG. 6. Normal liver parenchymal cell from untreated group. Prominent in this figure are the nucleus (N), nucleolus (Nuc), mitochondria (M) and glycogen masses (gl). x 10,700.
FIG. 7. Aflatoxin treated hepatic cell with dilated cisternae (c). The vacuolar distensions in the cytoplasm are oval and relatively uniform in size, x 19,600.
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on June 9, 2016
FIG. 4. Pancreas specimen from aflatoxin group. High magnification of virus particles. The dense central nucleoid is surrounded by a material of lower density and surface projections are present on the outer membrane, x 108.800.
C. Liver. In the aflatoxin-treated group. changes were detected both in individual cells and in tissue organization. While cisternae
321
CHRONIC AFLATOXICOSIS
FIG. 8. Liver from aflatoxin-treated bird showing numerous large vacuoles (Vac), x 12,200.
FIG. 9. Liver tissue of aflatoxin-treated group showing bile ductule. Three cells and part of a fourth comprise this section of the ductule, x 14,500. concomitant increase in connective tissue nuclei and collagen fibrils. Collagen bundles were found in the livers of some treated birds. Large numbers of heterophils, lymphocytes, and plasma cells were present in liver of all treated birds. DISCUSSION In heart tissue, the most outstanding ultrastructural changes were observed in mitochondria. The lipid-like mitochondrial inclusions were similar to inclusions described in human liver mitochondria by Rouiller and Jezequel (1963), who concluded that the droplets were lipid. The intramitochondrial vacuoles observed (Fig. 3) were similar to the membrane-limited matrix cavities of mitochondria found in rat liver 24 hours following aflatoxin administration (Svoboda et al., 1966). The presence of lipid inclusions in mitochondria could reflect general disturbance in lipid metabolism, but this would appear unlikely since the inclusions were not found in pancreas and liver. This is possibly related to the intense respiratory activity of heart mitochondria. The virions observed in the heart of one aflatoxin-treated bird were also found in the pancreas and liver of the same bird (Fig. 4). The virus particles resemble the leukosis
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on June 9, 2016
of the ER in the control group were not visibly dilated (Fig. 6), all of the treated group tissue had some degree of cisternal dilation (Fig. 7). There were a few ribosomes adherent to the cisternal membranes but there was no continuous ribosomal lining such as is found for control ER. Parenchymal hepatic cells of the aflatoxintreated group had large vacuoles which were not membrane-bound and of extreme size variation (Fig. 8). Some of the moderate sized vacuoles appeared to coalesce to form large vacuoles. Intranuclear change was minimal. Nucleolar segregation was occasionally seen in aflatoxin-treated livers, but not as frequently as in the pancreas. The most striking changes in aflatoxintreated liver were in tissue organization. Bile duct proliferation, the most common lesion in acute aflatoxicosis, was confirmed for chickens in this study. In some areas of the treated tissues, proliferating ductules were surrounded by connective tissue and cells of inflammation. Usually the ductules were composed of three or more cells joined to form a lumen (Fig. 9). In areas where normal tissue organization had been lost, bile duct epithelial cells could be seen alone or in pairs forming incomplete ductules. There was a vast increase in inflammatory cells with a
322
V. C. KELLEY AND E. C. MORA
of Pong and Wogan (1970), who found that nucleolar segregation and RNA polymerase inhibition, observed up to 12 hr. following administration of aflatoxin, had disappeared within 36 hr. In the present study, segregated nucleoli were observed much less frequently in liver than in pancreas. Since this was a chronic feeding study, the presence of segregated nucleoli would not be expected. Their presence in pancreas, which is otherwise changed little, suggests that pancreas may exhibit some of the basic liver alterations but on a much slower timetable. It might then prove to be a good model tissue to determine the sequence of ultrastructural changes. Like nucleolar segregation, dilation of the ER cisternae is an early change of aflatoxicosis. In the aflatoxin-treated chickens of this study, cisternal dilations were observed in the ER of liver and pancreas, with the hepatic dilations being larger, more numerous, and more homogeneous (Fig. 7). While degranulation of rough ER was not noticeable in the pancreas, cisternae of liver cells were membrane bound and few if any ribosomes were attached. Butler (1966) found cisternal dilation to be the earliest change in rat liver. Similar results were obtained by Svoboda et al. (1966) with rats and by Theron (1965) with ducklings. Increased prominence of the smooth ER was a change consistently observed in the liver of rats in a chronic feeding study (Svoboda et al., 1966). Otherwise dilation of cisternae of ER has been associated with acute cytoplasmic changes. The cytoplasmic changes related to RNA and protein synthesis are believed to occur following a primary nuclear alteration (Svoboda and Higginson, 1968). This was consistent with the lesser degree of cytoplasmic change observed in pancreas, which was still in a stage of primarily nuclear change. In accord with the general finding that liver is the primary site of damage in aflatoxin poisoning of several species, the most exten-
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on June 9, 2016
viruses, a group of RNA viruses originally discovered in chickens as a cause of leukemias and solid tumors. There are at least three possible explanations for the presence of these viruses in the aflatoxin-treated chicken. First, their occurence in an aflatoxin-treated bird may be coincidental, since these leukoviruses have been found in apparently healthy chickens (Temin, 1972). Second, the viruses may have reached detectable levels within treated birds due to a decrease in immunity to microorganisms; this would correlate with a reported decrease in immunity to fowl cholera (Pier et al., 1970), Marek's disease, and coccidiosis (Edds et al., 1973) attributable to aflatoxicosis. A third possibility is induction of a latent avian leukovirus by aflatoxin. Such a phenomenon is not unknown, since chemical carcinogens have been shown to induce avian leukoviruses in normal chicken cells (Weiss et al., 1971). In the present study, there were no macroscopic changes in pancreas (Rivelli-Florentin, 1969) and pancreatic damage reported by others working with aflatoxin has not been severe (Asplin and Carnaghan, 1961; Diener et al., 1963). The most prominent change in pancreatic acinar cells of aflatoxin-treated chickens was nuclear segregation or "capping" (Fig. 5), a phenomenon shown by time-lapse photography to be due to a redistribution of the morphological components of the nucleolus (Reynolds et al., 1963). Nucleolar segregation into granular and fibrillar components has been a frequent change induced in liver by aflatoxin and several other chemical carcinogens (Svoboda and Higginson, 1968); it has been related to the inhibition of DNA-dependent RNA-polymerase (Pong and Wogan, 1970). The separation was found to be one of the immediate ultrastructural changes following administration of aflatoxin, but was not associated in studies of chronic aflatoxicosis in liver (Butler, 1966; Svoboda et al, 1966). Those observations are in agreement with the studies
323
CHRONIC AFLATOXICOSIS
but not in the later stages. This suggested that destruction was less advanced in those specimens with the complete ductules. In many areas of the aflatoxin-treated livers, parenchymal cells had been almost completely replaced by invading mononuclear and polymorphonuclear cells. This was similar to the conditions described by Carnaghan et al. (1966), in which islands of lymphocytic hyperplasia surrounded areas of degenerating duct cells. Normal parenchymal cells could be found in a few areas but these usually were not invaded by inflammatory cells, presenting the possibility that these might be areas of regenerating parenchymal cells. The small bundles of collagen observed in liver confirm previous observations (Goldblatt, 1969) of a mild to moderate fibrosis in turkeys, chickens, ducklings, and other animals. Extravascular erythrocytes were found only in areas of extensive tissue damage. In no case was there evidence of ultrastructural abnormalities in organelles that were in contact with erythrocytes. This was in contrast to the report of Theron (1965), who found, in acute toxicity studies, that organelles in contact with extravascular erythrocytes were specifically altered. Other workers (Svoboda et al., 1966; Butler, 1966) have not observed this phenomenon. REFERENCES Asplin, F. D., and R. B. A. Carnaghan, 1961. The toxicity of certain groundnut meals for poultry with special reference to their effect on ducklings and chickens. Vet. Rec. 73: 1215-1218. Barnes, J. M., 1967. Toxic fungi with special reference to aflatoxin. Trop. Sci. 9: 64-74. Butler, W. H., 1964. Acute liver injury in ducklings as a result of aflatoxin poisoning. J. Path. Bacteriol. 88: 189-196. Butler, W. H., 1966. Early hepatic parenchymal changes induced in the rat by aflatoxin B,. Amer. J. Pathol. 49: 113-128. Carnaghan, R. B. A., 1967. Hepatic tumors and other
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on June 9, 2016
sive pathological changes observed in this study were in the liver. Livers from chickens on an aflatoxin diet all had some degree of vacuolation of the parenchymal cells (Fig. 8). Vacuolation of liver parenchymal cells has been found to be associated with aflatoxicosis of turkeys (Wannop, 1961), ducklings (Madhaven and Rao, 1966), and chickens (Carnaghan et al., 1966). In this study, no mitochondrial abnormalities of liver and pancreas were seen. However, in the hearts of the same animals, there were dramatic mitochondrial aberrations. Early mitochondrial changes in hepatocytes have been reported in aflatoxin poisoning of rats and ducklings (Butler, 1966; Theron, 1965) but have not been a feature of chronic toxicity studies. These data suggest that mitochondria are affected only secondarily and can recover. Possibly, toxicity to pancreas was too slight to be reflected in mitochondrial abnormalities. The pattern of bile duct epithelial proliferation in chicken was similar to that in duckling (Butler, 1964) and swine (Loosmore and Harding, 1961). Studies of Rhode Island Red chicks fed rations containing 1500 p.p.b. of aflatoxin B , showed slight proliferation of bile duct epithelial cells within 3-1/2 days (Carnaghan et al., 1966). Within 2 weeks, bile duct proliferation was prominent and increased through the sixth week. Because the level of aflatoxin used by Carnaghan was nearly four times that used in this study, the degree of proliferation of bile duct epithelium reported here after 8 weeks possibly does not represent the maximal possible proliferation. In only one treated chicken were complete ductules numerous (Fig. 9). In all treated groups, single cells tentatively identified as bile duct epithelium or, less frequently, pairs of ductular cells forming incomplete ductules were numerous in damaged areas. Butler (1964) observed the formation of complete ductules in the early stages of aflatoxicosis
324
V. C. K E L L E Y AND E . C.
Reynolds, R. C , P. O.' B. Montgomery and D. H. Karney, 1963. Nucleolar "caps"-a morphologic entity produced by the carcinogen 4-nitroquinoline N-oxide. Cancer Res. 23: 535-538. Rivelli-Florentin, E., 1969. The effects of aflatoxin on different breeds and strains of chickens. M.S. Thesis, Auburn Univ., Auburn, Ala. Rivelli-Florentin, E., G. J. Cottier, U. L. Diener and N. D. Davis. 1969. The effect of aflatoxin on different breeds and crosses of chickens. Poultry Sci. 48: 1809. Rouiller, Ch., and A. M. Jezequil, 1963. Electron microscopy of the liver, p. 195-264. In: The Liver, Ch. Rouiller (ed.), vol. 1. Academic Press, New York. Svoboda, D. J., H. J. Grady and J. Higginson, 1966. Aflatoxin B, injury in rat and monkey liver. Amer. J. Pathol. 49: 1023-1051. Svoboda, D., and J. Higginson, 1968. A comparison of ultrastructural changes in rat liver due to chemical carcinogens. Cancer Res. 28: 1703-1733. Theron, J. J., 1965. Acute liver injury in ducklings as a result of aflatoxin poisoning. Lab. Invest. 14: 1586-1603. Temin, H., 1972. The RNA tumor viruses. Background and foreground. Proc. Nat. Acad. Sci. 69: 10161020. Wannop, C. C , 1961. The histopathology of turkey " X " disease in Great Britain. Avian Dis. 5: 371-381. Weiss, R. A., R. R. Friis, R. Katz and P. K. Vogt, 1971. Induction of avian tumor viruses in normal cells by physical and chemical carcinogens. Virology, 46: 920-938. Wogan, G. N., 1974. Biochemical effects of aflatoxins. Israel J. Med. Sci. 10: 441-445. Wogan, G. N., and P. M. Newberne, 1967. Dose-response characteristics of Aflatoxin B carcinogenesis in the rat. Cancer Res. 27: 2370-2376.
NEWS AND NOTES (Continued from page 295) Huck has assumed overall direction for the Company's pharmaceutical and animal health divisions in the United States and Canada, its industrial and environmental health businesses and its consumer products business. Thomas B. Davis has been elected Vice President and General Manager of the Division that retains the Merck Chemical Division designation. It was formerly
the industrial and fine chemicals area. Spencer A. Stouffer has been elected Vice President and General Manager of Merck Chemical Manufacturing Division, formerly the operations area. ELANCO NOTES Robert E. Howerton has been promoted to Executive Director, Marketing-Animal Products for Elanco
(Continued on page 340)
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on June 9, 2016
chronic liver changes in rats following a single oral administration of aflatoxin. Brit. J. Cancer, 28: 811-814. Carnaghan, R. B. A., G. Lewis, D. S. P. Patterson and R. Allcroft, 1966. Biochemical and pathological aspects of groundnut poisoning in chickens. Pathol. Vet. 3: 601-615. Diener, U. L., N. D. Davis, W. D. Salmon and C. O. Prickett, 1963. Toxin-producing Aspergillis isolated from domestic peanuts. Science, 142: 14911492. Edds, G. T., K. P. C. Nair and C. F. Simpson, 1973. Effect of aflatoxin B, on resistance in poultry against cecal coccidiosis and Marek's disease. Amer. J. Vet. Res. 34: 819-826. Goldblatt, L. A., 1969. Aflatoxin. Academic Press, New York. Hamilton, P. B., 1971. A natural and severe occurrence of aflatoxicosis in laying hens. Poultry Sci. 50: 1880-1882. Kratzer, F. H., D. Bandy, M. Wiley and A. N. Booth. 1969. Aflatoxin effects in poultry. Proc. Soc. Exptl. Biol. Med. 131: 1281-1284. Loosmore, R. M., and J. D. J. Harding, 1961. A toxic factor in Brazilian groundnut meal causing liver damage in pigs. Vet. Rec. 73: 1362-1364. Madhaven.T. V., andK. Rao, 1966. Hepatic infarction in ducklings in aflatoxin poisoning. Arch. Pathol. 81:520-524. Pier, A. C , and K. L. Heddleston, 1970. The effect of aflatoxin on immunity in turkeys to bacterial challenge. Avian Dis. 14: 797-809. Pong, R. S., and G. N. Wogan, 1970. Time course and dose-response characteristics of aflatoxin B, effects on rat liver RNA polymerase and ultrastructure. Cancer Res. 30: 294-304. Pons, W. A., and L. A. Goldblatt, 1965. The determination of aflatoxins in cottonseed products. J. Amer. Oil. Chem. Soc. 42: 471-475.
MORA
