Responses of Dairy Calves to Oral Doses of Aflatoxin

Responses of Dairy Calves to Oral Doses of Aflatoxin G. P. LYNCH, W. T. SHALKOP,I N. M. JACOBY, D. F. SMITH, and R. W. MILLER Animal Science Research Division, ARS, USDA Beltsville, Maryland 20705 Abstract

Seven pairs of young male dairy calves received daily oral doses of aflatoxin from 0 to .10 m g / k g body weight for 6 weeks. The experiment was divided into 6 weeks of pretreatment and 6 weeks of aflatoxin dosing. Analysis was by least squares analysis of variance. Differences (P < .01) for dose and dose X period interaction were shown for a reduction of feed intake, weekly gain, serum carotene, and for an increase of serum alkaline phosphatase. Differences (P < .01) for dose X period interaction were also shown for an increase of total bilirubin and a decrease of serum inorganic phosphorus. Serum vitamin A was decreased (P < .05) by dosing with a (P < .05) dose X period interaction. Most of these aflatoxininduced effects occurred by the second week of treatment and at .08 and .10 mg doses. A t postmortem livers appeared light tan to orange and gall bladders were enlarged to 10 times normal. Liver tissue showed bile duct proliferation at .04 mg and above. Fibroblastic proliferation, dilated intralobular lymphatic ducts, perivascular edema, and edema around the neck of the gall bladders was evident. Histochemieal determination of alkaline phosphatase was not indicative of hepatic cell damage or obstructive jaundice. At aflatoxin doses .08 and .10 mg, glycogen deposits appeared only in regenerating liver cells. Responses of young male dairy canes to aflatoxin contaminated feed and oral doses of crude toxin are similar.

of Aspergillus flavus (1, 2), and isolates of other fungal species (3). Previous work (11) with feeding aflatoxin contaminated feed to young dairy calves has shown pathological and clinical biochemical changes similar to those in other mammalian species. The liver of the calf is one of the first organs affected by aflatoxin. As time progresses with dairy canes under aflatoxin treatment, feed intake is reduced and physiological responses due to afiatoxin treatment and reduction in feed intake become difficult to separate. Further work is needed to help identify specific pathological and biochemical changes that can be attributed to aflatoxin treatment. The sequence of these changes with time would also be of value in evaluating the total effect of aflatoxin on the calf. The purpose of this experiment was to evaluate clinical biochemical, and pathological lesions produced with time by daily oral administration of aflatoxin to young male dairy calves. Experimental Procedure

Six dose levels of aflatoxin were administered orally to six pairs of 4 to 5 month old male dairy calves for 6 weeks. A seventh pair of TABLE :1. Average initial and final body weights, doses and the average total amount of aflatoxin administered orally to pairs of dairy calves.

Pair

Initial weight

Final weight

(kg) 96.3 80.0 83.6 75.4 84.3 67.5

(mg/kg body wt.) .00 .02 .04 .05 .06 .08

66.5

.10

Introduction

Aflatoxin is a general name of a group of toxic, fungal metabolites containing a basic coumarin ring and produced by some strains Received for publication April 9, 1971. 1 Veterinary Pathologist, Laboratory Division, Consumer & Marketing Service, USDA, ]~eltsville, Maryland 20705. 1688

1 2 3 4 5 6 7

(kg) 50.3 44.1 46.4 43.0 50.0 44.6 46.8

Experimental dose

Total aflatoxin administered (avg mg) 00.0 57.7 123.6 138.4 183.5 217.1 280.1

1689

A F L A T O X I N E F F E C T S ON CALVES

calves was designated as controls aud received no aflatoxin. Aflatoxin doses were on the basis of milligrams total aflatoxin per kilogram body weight per day. These doses ranged from .02 to .10 m g / k g (Table 1) with weekly correction of doses for changes in body weight. The oral dosing solutioa was prepared by dissolving aflatoxin crude powder s in an equal mixture (v:v) of propylene glycol and 95% ethanol. Each daily dose was administered in a gelatin capsule. The experiment was divided into two phases; a 6 week pretoxin period and 6-week toxin dosing period. 2 Aflatoxin crude powder was supplied through the courtesy of P. Rogovin, Northern Utilization Research and Development Division, USDA, Peoria, Illinois. Analyses for all batches were B1, 52.6 to 65.1%; G1, 8.9 to 9.8%; Be, 9.5%; and G~, .6 to 6.1%.

All calves received 3 liters of whole milk daily, plus sufficient quantities of hay and a 12% protein grain ration s for a growth of 2 to 3 kg weekly. During the experiment, feed intakes and body weights were recorded. Weekly, 12-hr fasted, venous blood samples were taken for various clinical biochemical assays. These determinations included serum alkaline phosphatase (AP) (10), serum ornithine carbamyl transferase, (OCT) (13), serum albumin:globulin ratio ( A / G ratio) by electrophoresis with cellulose polyacetate as the supporting phase and a Tris-barbital-sodium barbital buffer ( p H 8.8, ionic strength .05) as the liquid phase, total serum protein by the biuret method (6), serum glucose by the glu3 Corn 352 parts, whole oats 352 parts, linseed meal 60 parts, wheat bran 180 parts, trace mineral salt 6 parts, and molasses 50 parts.

pl PR]FI WU.] PR]R ND. 2 7

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~

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~EEK.~ Fro. I. Least squares means of feed intake plotted by dose for the toxin dosing period. Pairs of calves are numbered by increasing aflatoxin dose. Pair 7 is the control. JOURNAL OY DAIRY SCIENCE ~0~. 54, NO. 11

0

TABLE 2. Analysis of variance and mean squares for variables of calves dosed with aflatoxin. o

Source of ~ variance

A/G

df

Feed intake

Body wt gains

Dose Linear Quadratic Higher

6 1 1 4

28.16** 74.09** 34.24 ~* 15.16 ~'°*

10.60"* 56.54** 2.54 ~ 4.54 *~

644.79** 2792.33 ¢'* 207.73 217.17

787613.0 48353.7 2790441.1 471720.7

.82 .99 2.47 .36

563.79 28.81 33.18 830.18

Animal/dose '~

7

.65

.37

55.08

835349.3

.73

274.94*

1

99.21 *~

Period* 9 ~, Dose X period ~a

8.06

AP

2078.35 ~

OCT

3067742.6 ~'~*

ratio

.92**

Glucose

38.10

439.98**

151635.5

.25

440.61"*

1.70

17.68 ~*

44069.6

.06

44.11

6.38"* 29.31"* 2.20** .14

6.20"* .02 2.39 26.20**

126.97"* 522.63 *~ 33.23 * 26.32 *°'~

169645.7** 608890.9 52924.6 62137.6

.I0" .06 .02 .14"

168.50 ~* 9.72 727.41"* 35.13

30 1 1 1 1 26

1.62"* 22.37** 6.34** 6.78"* .73" .47"*

2.75** 3.42 .97 1.05 7.28* 2.69*

57.48 ~* 675.29** 120.36"* 91.21"* 231.23"* 23.32"*

51817.8 57981.2 6339.4 4.3 3158.3 57193.5

.07* .02 .09 .65** .05 .05

215.11"* 582.88** 764.71"* 356.48** 848.69** 150.02"*

5

10.51"*

6.79**

167.96"*

50576.4

.10

Dose X per. X wk.

30

2.12"

4.47**

52.00**

75707.0

.03

66.91"*

Error/animal

70

.11

5.78

48554.7

.04

23.86

6

32.64**

Period X an/dose b

7

.81"*

Weeks Linear Quadratic Higher

5 1 1 3

Dose X weeks Lin X lin. Lin X quad. Quad X lin. Quad. X quad. Higher Period X week

a Dose error. b Period error.

16.19 °~*

1.38

66.71"

AFLATOXIN

EFFECTS

cose oxidase method (16), plasma carotene and vitamin A by the Kimble method (9), serum bilirubin (8) and serum phosphorus by the molybdate method (15). A t termination of the 6-week toxin feeding, calves were sacrificed for gross postmortem observations, and tissue samples were removed for pathologic examination. Samples of each tissue were fixed in formalin, stained with hemotoxylin and eosin. Samples of tissues were also frozen, cut on a cryostat, and stained with oil red O for neutral fat and periodic Schiff reagent for determination of glycogen. Results and Discussion

The variance of measures of physiological response of dairy calves to aflatoxin treatment was analyzed by least squares. Means square and significance are shown for each variable,

1691

ON CALVES

Tables 2 and 3. Table 4 lists pretreatment means, treatment period means by dose and standard error for each variable tested. There was a dose effect (P < .01) and dose × period interaction for feed intake, body weight gains, serum AP, and serum carotene. A quadratic reduction (P < .01) in feed intake by weeks was apparent (Table 2) at .08 mg and .10 mg doses (Table 4) by the second week of treatment (Fig. 1). A decrease (P < .01) for a higher order reaction in rates o£ gain by weeks (Table 2) was also shown at .08 mg and .10 mg aflatoxin doses (Table 4). P r i o r work (11) has indicated no significant differences in rate of gain when aflatoxin contaminated feeds were fed to dairy calves. I n this experiment, aflatoxin dosing was independent of feed hltake allowing higher total intakes of aflatoxin. F o r example, in prior work

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b.a o >

o.

TABLE 3. A n a l y s i s of v a r i a n c e and m e a n squares f o r variables of calves dosed with aflatoxin. Source o f variation

df

.o

Carotene

Direct bilirubin

Total bilirubin

Phosphorus

Protein

6

110.88 ~

997238.6 **

.21

1.49 ~

9.85

3.74

Linear

1

35.50

74466.8 *

.64*

5.50 ~

11.22

8.67

Quadratic

1

206.21 *

445517.6 ~

.09

2.31 ~

16.14

1.34

Higher

4

105.90

119313.5 ~

.13

.28

7.93

3.11

A n i m a l / d o s ea

7

27.29

5450.3

.09

,23

3.75

4.24 *~

Period

1

177.53 ~

551542.3 *~

.28

3,43 e*

2.75

12.87 *~

6

73.68 ~

77589.4 ~'*

7

12.56 ~

4357,2 **

Dose

.~

Vitamin A

~, Dose × p e r i o d Period × an/dose D Weeks

.15 .09 ~

11,03 ~

14.50 *~

1.38 ~

.13

1.53 ~

.28

6.26 *~

.39

5

40.72 ~

1059.5

.08 *

.65 ~

Linear

1

48.94 ~*

162,9

.32 ~

2.66 ~"~

Quadratic

1

45.52 ~'~*

1677.0

.03

.06

.79

1.42

Higher

3

36.38 ~

1152.5

.02

.18

1.52

.16

Dose X weeks Lin × lin. L i n × quad. Quad. ×

quad.

Higher P e r i o d × week Dose × per. × week Error/animal

30

12.46 ~

4678.6 ~*

1

36.91 ~

23015.1 ~*

1 1

30.93 ~

1

2.04

26

11.66 ~*

236.0 62887.5 *~

.36 ** .01

.07

.20*

.58

3.06 ~*

6.08 ~

1.69

.00

.39

.74

1.20

.31

.61 ~

.07

1.07 ~

1475.8

.00

.00

.02

.75

2028.6

.03

.07

.37

.57

5

55.54 ~

34551.5 ~*

.I0 ~

.48 ~

1.10

.67

30

16.73 ~*

5233.5 ~*

.05

.20 ~"

1.43 ~*

.59 ~

1289.3

.03

.08

70 a Dose error. b P e r i o d error.

.00

.04

25.94 ~

5.03

.61

.37

>

AFLATOXIN EFFECTS

the 6 week toxin intake at .08 mg dose was 79 mg B1 whereas in this experiment it was 128 mg B 1 (assuming an aflatoxin B1 content of 59% for all batches). Total aflatoxin administered by dose is in Table 1. There was a linear increase (P < .01) of serum A P due to aflatoxin dose and a higher order reaction (P < .01) by weeks (Table 2). This increase in serum A P was also evident at .08 mg and .10 mg doses (Table 4) during the first to second week of treatment (Fig. 2). Table 3 shows a higher order effect (P < .01) for dose on serum carotene. Means in Table 4 indicate variability in decrease of serum carotene among increasing aflatoxin doses. Serum glucose was not significantly affected by dose, but there was a dose × period interaction (P < .01). A large variation in the serum glucose is indicated by the animal/dose response (P < .05) (Table 2). Post treatment

1693

ON C A L V E S

and least squares means for serum glucose are in Table 4. There was a dose effect (P < .05) and a dose × period interaction on plasma vitamin A (Table 3). A higher order decrease (P < .01) in plasma vitamin A_ by weeks is shown in Table 3. Quadratic effect of dose on total bilirubin (Table 3) was significant (P < .05) and the dose × period interaction significant ( P .01). A linear increase (P < .01) in total bilirubin by weeks (Table 3) was clearly evident at .08 mg and .10 mg doses (Table 4) by the second or third week of treatment

(Fig. 3). A dose × period interaction ( P < .01) was shown for serum phosphorus (Table 3). A linear reduction (P < .01) in serum phosphorus by weeks is shown (Table 3) at .08 mg and .10 mg doses (Table 4) by the third

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Fro. 3. Least squares means for total serum bilirubin plotted by dose for the toxin dosing period. JOURNAL OF DAIRY SCIENCE VOL. 54, NO. 11

I-a o

TABLE 4. Means and standard errors of variables from pretreatment and treatment periods of calves dosed with aflatoxin. .~

Feed intake

~. o

Body wt gains

AP (e.u.) a

OCT (e.u.) a

A/G ratio

Glucose (rag/ 100 ml)

--(kg/week)--

Direct ¥itamin billA Carotene rubin ~(t~g/g)--

Total billrubin

Phosphorus

Protein

(mg/lO0 m l ) - -

Pretreatment average

2.81

2.90

5.3

274

1.37

76.5

8.1

111

.15

.39

8.26

5.8

Treatment period Dose .00

5.91

4.25

3.5

210

.95

81.7

10.0

117

.08

.41

8.84

5.8

.02

5.44

3.14

6.2

708

1.11

72.8

6.8

106

.14

.26

7.86

6.4

.04

5.67

3.39

7.5

610

1.42

79.7

14.8

310

.16

.26

8.92

6.0

.05

5.44

2.61

9.3

852

1.39

75.8

11.5

353

.17

.48

8.73

6.2

.06

5.65

2,95

8.4

739

1.53

75.5

15.0

432

.18

.45

9.44

6.3

.08

1.03

.72

28.2

325

.96

73.8

6.7

117

.57

1.36

6.02

6.9

.10

1.30

.14

23.3

366

1.16

69.8

5.9

146

.33

1.49

6.21

6.6

.57

.43

5.2

646

.60

11.7

3.7

52

.22

.34

1.37

1.5

SE a Sigma enzyme units.

"~

1695

AFLATOXIN EFFECTS ON CALVES

week of treatment (Fig. 4). Serum protein showed a dose × period interaction (P < .05). A large variation in serum protein response due to aflatoxin treatment was indicated by an animal/dose interaction (P < .01) (Table 3). These results indicate several important physiological relationships in young dMry calves dosed with aflatoxin. Serum A P rises with aflatoxin treatment (2, 11). This is an early aflatoxin response and has been related to possible liberation of the enzyme from damaged hepatic cells. Cornelius (4) reports a wide range of normal values for serum A P among individual cattle and questions its use as an indicator of liver insufficiency or obstructive icterus. Our work with dairy calves indicates this is a consistent dose related response to afiatoxin poisoning. Increased OCT enzyme also indicates liver cell damage in mammals as

this enzyme is specifically located in liver tissue (13). Afiatoxin dosing of dairy calves produced no meaningful change in serum OCT (Table 2). Since afiatoxin inhibits protein synthesis, it was of interest to know what effect aflatoxin dosing would have on the & / G ratio and total serum protein. Statistical analysis (Tables 2 and 3) indicated no important change on A / G ratio due to treatment. These results with the A / G ratio were similar to those reported earlier (11). This indicates that the liver retained some of its functional ability to mainrain electrophoretie serum proteins during aflatoxin treatment. One of the many important functions of the liver is that of vitamin A storage. A reduction of liver vitamin A stores by aflatoxin has been shown previously (2). I n this study both serum vitamin A (Table 4) and vitamin A content of the liver at postmortem were reduced by

10.

~ 9.0

,

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o 8:0

=o o_

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HEEK5

1

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2

I

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FIG. 4. L e a s t s q u a r e s m e a n s o f s e r u m i n o r g a n i c period.

dosing

'1

NO, I NO.R NO,~ NB.; NO,5

phosphorus

plotted

by

dose

for

the

toxin

JOUR~AL OF DAIBY Sel~ev. VOL. 54, NO. 11

1696

LYNCH

ET AL.

TABLE 5. Average postmortem carotene and vitamin A in pairs of dairy calves dosed with aflatoxin. Aflatoxin dose (mg/kg body wt) .00 a

.02 .04 .O5 .06 .08 .10

Liver carotene

(~g/g) ...... 12.0 11.7 14.0 12.5 3.9 5.8

Liver vitamin A

(sg/g) 10.9 15.6 14.1 11.4 3.6 5.3

a Sample~ deshcyed. aflatoxin treatment. Table 5 shows a postmortem reduction in both liver carotene and vitamin A at .08 mg and .10 mg doses. Two gross symptoms appeared frequently and seemed to be associated with high aflatoxin doses. These were loss of fecal pigmentation and icteric sera. As a result of this, bilirubin determinations were made on weekly serum samples. A rise in total serum bilirubin was shown (Fig. 3). This clinical finding, along with the loss of fecal pigmentation, indicates an extrahcpatic biliary obstruction. Sermn inorganic phosphorus is related to glycogen utilization through phosphorolysis (5). Clinically, during an increase in carbohydrate utilization, serum inorganic phosphorus decreases (4). This observation in calves can be borne out by the fact that in this experiment

Fio. 5. Enlarged gall bladder removed from an aflatoxin treated calf at postmortem. Jov~ o r DAVY S O ~ C ~ ~roL. 54, NO. 11

FIG. 6. Hematoxylin- and eosin-stained section from an aflatoxin-dosed calf showing extensive bile duct hyperplasia (long arrows). Dilated intralobular lymphatic ducts are indicated (short arrow) at the periphery of the lobule. (× 100). serum phosphorus was reduced (Fig. 4). Prior work has indicated a loss of liver glycogen in histoehemical liver sections from aflatoxin treated calves (11). Pathological examination of the experimental calves at postmortem indicated some important aflatoxin-induced changes. The liver appeared to be a target organ and at the two highest aflatoxin levels was light tan to yellow. Similar results were shown in earlier work with calves (11). Gall bladders were enlarged from two to i 0 times their normal size (Fig. 5), with edema surrounding the neck of the bladders. Bile in all cases was normal in color and flowed freely upon incision of the gall bladder. On microscopic examination of liver sections, the most consistent findings were diffuse bile duct proliferation (Fig. 6) with accom-

FIG. 7. Section of liver from an attatoxlndosed calf stained with Gomori's reticulum stain showing extensive reticular proliferation and disorganization of the normal ]obular architecture. (× 100).

AFLATOXIN

EFFECTS

ON CALVES

1697

FIG. 8. Hematoxylin- and eosin-stained liver section from a calf dosed with aflatoxin showing portal triad with greatly distended lymphatics (arrows). (× 100).

FIG. 10. Frozen liver section from a control calf stained with the Gomori's cobalt technique for alkaline phosphatase showing normal enzyme activity in the bile ca~laliculi (arrows). (× 100).

partying fibroblastic proliferation (Fig. 7), greatly distended lymphatic ducts (Fig. 8), and perivascular edema of both arteries and veins (Fig. 9). The severity of these changes was dose related and first observed at .04 rag. The severity of these changes, at corresponding aflatoxin doses, appeared greater with t r a d e toxin than with aflatoxin-eontaminated feed in previous work (11). This may be related to the fact that a greater total intake of toxin was possible by the dosing procedure in this experiment. Histochemically, there was little indication of glycogen loss from liver cells up to .06 mg aflatoxin when bile duct and fibroblast proliferation was moderate. A t the two highest aflatoxin levels, .08 mg and .10 rag, there were only microfocal glycogen deposits, and these appeared in individual regenerating liver ceils.

Although both serum and liver vitamin A values were decreased, the deficiency period was apparently too short to produce consistent morphologie changes. F o r example, metaplasia was not present in kidney pelvic sections. I t has not been thoroughly established, in experimental work with laboratory animals, whether the increase in serum A P in hepatobiliary injury is the result of increased production of hepatic phosphatase or impaired clearance of extrahepatie phosphatase from the blood by liver cells. P o p p e r (12) suggests that increased A P activity can be attributed to both hepatic formation and, to a lesser degree, to piling up of extrahepatic phosphatase. I-Ie also states that hepatic phosphatase production appears to be stimulated more by cholestasis than by hepatic cell damage. Cholestasis

FIG. 9. Hematoxylin- and eosin-stained section from an afiatoxin-dosed calf showing marked perlvascular edema of the central vein (braces). Proliferation of the vein is also evident (arrow). (× 100).

FIG. 11. Frozen liver section from an aflatoxindosed calf stained with the Gomori's cobalt technique for alkine phosphatase showing disorganization of the hepatic plates and enzyme activity confined to areas of bile duet proliferation (arrows). (X 100). ~OURNAL OF DAIRY SCIENCE VOb. 54, NO. 11

1698

LYNCH

usually produces a backup of phosphatase in the canaliculi and can be demonstrated with histochenfical stains. I n this experiment the gross observation of biliary retention in the gall bladder and the loss of fecal pigmentation supports an extrahepatic obstructive type of icterus (4) with consequent cholestasis. ~Iowever, livers f r o m calves with the extrahepatic obstruction did not contain increased amounts of A P in the bile canaliculi (Fig. 10 and 11), although small plugs of A P were related to p r o l i f e r a t i n g bile ductules. There was no phosphatase reaction in new connective tissue accompanying bile duct p r o l i f e r a t i o n or evidence of its release f r o m injured liver cells. Therefore, histochemical stains for liver A P in these calves were inconsistent both as an explanation for the source of increased serum A P and as an indicator of an obstructive t y p e of jaundice. The high serum A P , on the other hand, was entirely consistent with the gross findings of extrahepatie biliary obstruction (12). I n p r i o r work with aflatoxicosis in the p i g ( ] 4 ) , it was not unusual to find the gall bladder atrophic and the wail edematous. I n calves on higher aflatoxin, the area surrounding the neck of the gall bladder was edematous, but the bladders were greatly extended with bile. I n the bovine, the hepatic duet and common bile duct have thick circular and longitudinal muscle layers, which are not found in other domestic animals (7). The marked retention of bile in the gall bladder of calves m a y be explained by either interstitial edema with consequent compression at the periductal structures, or the edema may have produced a functional i m p a i r m e n t of the muscle layers, extending to sphincter, so that the latter did not respond to the relaxant hormone cholecystokinin. W h e n y o u n g dairy calves are dosed with a p r e p a r a t i o n of crude aflatoxin, marked physiological responses to the toxin are shown as early as the second week of treatment. Pathological changes were noted at some o f the lower doses, e.g., bile duet h y p e r p l a s i a at .04 rag. M a n y clinical biochemical changes were found consistently at .08 mg and .10 mg aflatoxin. This may be interpreted to mean that tissue changes may precede clinical biochemical changes. Most physiological changes in this experiment app e a r to be associated with functions of the liver, but possible aflatoxin effeets on funetions of other organs and tissues should not be overlooked.

Acknowledgments The authors wish to thank Dr. Richard P. Lehman, Food and Drug Administration and Dr. JOURNAL OF DAIRY SCIENCE VOL. 54, NO. 11

ET AL.

B. T. Weinland, Biometrical Services, ARS, USDA, for assistance in the statistical design and analysis of this experiment.

References (I) Allcroft, R., and R. B. A. Carnaghan. 1963. Toxic products in groundnuts. Biological effects. Chem. Ind. (London), p. 50. (2) Allcroft, R., and G. Lewis. 1963. Groundnut toxicity in cattle: experimental poisoning of calves and a report on clinical effects in older cattle. Vet. Rec., 75: 487. (3) Allcroft, R. 1965. Aspects of aflatoxicosis in farm animals. I n Mycotoxins in Foodstuffs, ed. G. N. Wogan, MIT Press, Cambridge, Massachusetts. (4) Cornelius, C. E., and J. J. I~[aneko. 1963. Clinical Biochemistry of Domestic Animals. Academic Press, New York. (5) Fruton, J. S., and S. Simmonds. 1958. General Biochemistry, 2nd ed., John Wiley and Sons, Inc., New York. (6) Gornall, A. G., C. J. Burdawill, and M. M. David. 1949. Determination of serum proreins by means of the biuret reaction. J. Biol. Chem., 177: 751. (7) Habel, R. E., and E. L. Biberstein. 1952. The IIistoIogy of Domestic Animals, Comstoek Pub. Associates, Ithaca, New York. (8) Jendrassick, L., and P. Grof. 1938. Vereinfaehte photometrische methoden zur bestimmung des blutbilirubins. Biochem. Z., 297: 81. (9) Kimble, M. S. ]939. Photo-colorimetric determination of vitamin A and earotene in human plasma. J. Lab. Clin. Med., 24: 1055. (10) King, E. J., and A. R. Armstrong. 1934. Convenient method for determining serum and bile phosphatase activity. Canadian M. A. J., 31: 376. (11) Lynch, G. P., G. C. Todd, W. T. Shalkop, and L. A. Moore. 1970. Responses of dairy calves to aflatoxin contaminated feed. J. Dairy Sci., 53: 63. (12) Popper, H., and F. Schaffner. 1957. Liver: Structure and Function. McGraw-Hill, New York. (13) Reichard, H., and P. Reiehard. 1958. Determination of ornithine carbamyl transferaso in serum. J. Lab. and Clin. Med., 52: 709. (14) Shalkop, W. T. 1967. Proc. 71st Annu. Meet. U.S. Livestock Sanitary Ass., p. 265. October. (15) Taussky, It. H., and E. Shorr. 1953. A microeolorimetric method for the determination of inorganic phosphorus. J. Biol. Chem., 202 : 675. (16) Teller, J. D. 1956. Direct, quantitative, colorimetric determination of serum or plasma glucose. Abst. Papers, 130th Meeting, September, Amer. Chem. Soc., p. 69c.