EFFECTS OF AFLATOXINS ON FINISHING SWINE 1

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1 EFFECTS OF AFLATOXINS ON FINISHING SWINE 1 L. L. Southern 2 and A. J. Clawson 3,4 Nortb Carolina State University, Raleigh Summary Thirty-two pigs (average starting weight 53 kg) were randomly divided into four treatments with eight pigs per treatment. Fortified cornsoybean meal diets containing 14% protein were used. The diets contained 20, 385, 750 and 1,480 ppb total aflatoxin, respectively. Treatment one served as the control. Average daily gain was reduced linearly in pigs receiving diets containing 385 ppb and higher levels. Feed efficiency was not significantly influenced by dietary levels of 385 and 750 ppb aflatoxin, but was depressed when the highest level (1,480 ppb) was fed. Total serum proteins, albumin and IgG fraction (gamma globulins) were unaffected by aflatoxin consumption. IgM fraction (gamma globulins) was increased in pigs receiving 750 and 1,480 ppb aflatoxin. Liver weights expressed as a percentage of final live weight were increased in pigs that consumed 385 ppb and in each subsequent treatment. Microscopic examination of liver sections revealed that five of eight pigs that had received the highest level of aflatoxin had hepatocellular lesions. Four of eight pigs in treatment three, two of eight pigs in treatment two and one of eight pigs in treatment one also exhibited hepatocellular lesions. The remaining animals had no visible lesions. At the end of the 66-day feeding trial, l Paper No of the Journal Series of the North Carolina Agr. Res. Service, Raleigh. Use of trade names in this publication does not imply endorsement of the products mentioned. 2Present address: Animal Science Dept., Univ. of IL, Urb ana-champaign. 3 Animal Science Department. 4Acknowledgment is given to Dr. R. C. Dillman for his microscopic examination of liver sections, Dr. G. W. Morgan for his assistance with the electrophoresis and the North Carolina Department of Agriculture Food and Drug Division for their assistance in the analyses of corn for aflatoxin. one-half of the pigs were used in a short (7-day) withdrawal trial. The pigs placed on control diets consumed more feed, gained faster and were more efficient than the pigs that remained on their respective aflatoxin-containing diets. (Key Words: Aflatoxicosis, Swine, Total Serum Protein, Gamma Globulins, IgG, IgM, Growth, Feed/Gain.) Introduction Aflatoxicosis is one of the more serious problems affecting the livestock industry in the Southeastern United States. Smith et al. (1976) reported a survey of animals (swine, cattle and poultry) submitted to the North Carolina Diagnostic Laboratory for suspected aflatoxicosis. The overall incidence of confirmed aflatoxicosis was 25% of the animals submitted that were suspect. Swine Accounted for 88% of the confirmed cases. The feeds being consumed by these animals prior to submission were analyzed and found to contain a mean level of 3,890 ppb aflatoxin. Smith et al. (1976) concluded that aflatoxicosis was more severe than had been suspected. Numerous workers (Duthie et al., 1966, 1968; Hintz et al. 1967; Monegue et al., 1977 a,b) have induced experimental aflatoxicosis in finishing swine; unfortunately, the results are not in agreement, and a tolerance level could not be accurately estimated from the reports in the literature. The present experiment was designed to further evaluate the level of aflatox- in that can be ingested by finishing swine without altering performance or inducing clinical pathological signs of aflatoxicosis. The effect of aflatoxin on liver weight and serum proteins was determined and a histopathological examination of the livers was made. Experimental Procedure Thirty-two crossbred pigs were randomly divided into four treatment groups with two pens of four pigs each per treatment. The 1006 JOURNAL OF ANIMAL SCIENCE, Vol. 49, No. 4, 1979

2 EFFECTS OF AFLATOXINS ON FINISHING SWINE 1007 average initial weight of the pigs in the two replications was 50 and 55 kilograms. Their shelter was a completely enclosed building with curtain on one side. The pigs in one replication were housed in pens with totally slatted floors and the other in pens with partially slatted concrete floors. Water was furnished by automatic waterers and the feed provided ad libitum in self-feeders. Pig weights and feed consumption were obtained weekly during the 66-day experiment. One pig from treatment four was discovered in a moribund state on day 35. Necropsy revealed gastric hemorrhage as the cause of death. Data from this pig was deleted in the statistical analysis. A fortified 14% crude protein, corn-soybean meal diet (table 1) was used. Two different corn sources, designated control corn and ariatoxin corn, were used in preparing the diets. The control corn and ariatoxin corn were naturally contaminated with 25 and 1,800 ppb total aflatoxin, respectively. Three of the four major aflatoxins produced by Aspergillus flavus were present in the aflatoxin contaminated corn. The 1,800 ppb aflatoxin present consisted of 1,330 B1, 135 B2, 335 G1 and 0 ppb G2. The corn was analyzed for ariatoxin content with procedures outlined in AOAC (1975). Diets one and four contained only control corn and aflatoxin contaminated corn, respectively. Graded levels of aflatoxin were achieved for treatments two and three by blending control corn with aflatoxin contaminated corn. Dietary levels of aflatoxin were 20, 385,750 and 1,480 ppb for treatments one, two, three and four, respectively. Rectal temperatures of pigs that consumed diets one and four were taken on days 7, 14 and 21 of the experiment. On day 35, all pigs were bled from the anterior vena cava. The blood was maintained at room temperature for 4 hr and then centrifuged for 20 rain at 2,000 rpm. The serum was collected and stored at -18C until analyzed. Total serum protein was determined by the method of Lowry et al 1951) and the serum protein components were separated by disc gel electrophoresis (Davis 1964). At the end of the 66-day feeding trial, one-half of the pigs from each pen were placed on control diets and used in a 7-day withdrawal study. At the end of the withdrawal period, all the animals were slaughtered and their livers removed for analysis. The livers were weighed and a section removed from the left medial lobe TABLE 1. PERCENTAGE COMPOSITION OF DIETS lnternat'l Item Ref. No. Percent Control corn, grain, grnd. a (8.6) b Aflatoxin corn, grain, 82.4 grnd. a (8.6) b Soybean meal, dehulled solv. extd. (48.0) b Trace mineral salt c.5 Vitamin premix d.5 Dicalcium phosphate 6-O Ground limestone Selenium premix e.05 acontrol corn and aflatoxin corn were naturally contaminated with 25 and 1,800 ppb aflatoxin respectively. Aflatoxin levels for diets two and three were obtained by blending control corn and aflatoxin corn at ratios of 2:1 and 1:1 respectively. Diets one and four contained control corn and aflatoxin corn, respectively. The final dietary levels were diluted by approximately 18% as a result of adding soybean meal and minerals to the diets. bfigures in parentheses represent the protein content (N 6.25) expressed as a percentage of air-dried feedstuff. CTrace mineralized salt premix provided the following per kilogram of diet: sodium chloride, 4.7 to 4.9 g; zinc, 50 mg; manganese, 27.5 mg; iron, 4 to 15 mg; copper, 3 rag; cobalt,.75 mg; iodine,.65 mg. Diamond Crystal Salt Co., St. Clair, MI. dvitamin premix provided the following per kilogram of diet: vitamin A, 4,400 IU; vitamin D, 275 IU; vitamin E, 5.5 IU; vitamin B mg; riboflavin, 1.1 mg; niacin, 16.5 rag; d-pantothenic acid, 5.5 rag; choline chloride, 440 rag; Roche Vitamin Supply, Roche Chemical Division, Hoffman-La Roche, Inc., Nutley, NJ. eseleninm premix provide.1 mg per kilogram. of each liver and fixed in a 10% formalin solution for microscopic examination. All data were analyzed by the method of least squares (Barr and Goodnight 1972). Results Average daily gain (ADG) and average daily feed (ADF) consumption were lineraly reduced (P<.05) as level of dietary aflatoxin was increased (table 2, figure 1). The feed efficiency (F/G) was affected (P<.05) only by the highest level of aflatoxin (figure 2). Pigs that consumed the control diet during the 7-day withdrawal period consumed more feed (2.18 vs 2.05 kg/day) gained at a faster rate (.59 vs.30

3 1008 SOUTHERN AND CLAWSON TABLE 2. EFFECTS OF GRADED LEVELS OF AFLATOXIN ON FINISHING SWINE Performance Dietary aflatoxin level (ppb) a criterion b S E c ADG,dkg.77 e t f f g ADF, kg 2.87 e 2.53 f 2.15g 1.61 h 1i7 F/G ratio 3.74 e 3.78 e 3.71 e 3.97 f.08 aeach treatment contained two replications of four pigs each, average initial weight of the two replications was 50 and 55 kilograms. bmeans for seven pigs only. Cstandard error of the mean, SD/~N. dthe standard error of the mean is given for dietary aflatoxin levels. e'f'g'hmeans with the same superscript are not significantly different (P>.05). kg/day) (P<.01) and were more efficient (3.66 vs 6.91 kg feed/kg gain) than the pigs remaining on their respective aflatoxin containing diets. Rectal temperatures were apparently not affected by dietary treatment. Mean temperatures combined for days 7, 14 and 21 were and C, respectively, for pigs on treatments one and four. Total serum protein was not affected (P >.10) by level of aflatoxin consumed (table 3). There was an increase (P<.05) in the IgM fraction of the gamma globulins of pigs in treatments three and four when compared to pigs fed the control diet. The IgM levels ob- C---'O TREATMENT t H TREATMENT 2 I00 ~ TREATMENT 3 H TREATMENT 4 served in pigs fed diet four were increased (P<.05) over those fed diet two, but treatment two did not differ from treatments one or three. The IgG fraction of the gamma globulins and the albumin concentrations were unaffected (P>.10) by aflatoxin consumption (table 3). Liver weight expressed as a percentage of the final pig weight increased (P<.05) as dietary aflatoxin level increased (table 4). Microscopic examination of liver sections revealed that five of the remaining seven pigs in treatment four exhibited visible lesions (VL), bile duct proliferation or hyperplasia and (or) hepatocellular swelling, disorganization, megalocytosis and necrosis. Four of the eight pigs in treatment three were similarly diagnosed; two pigs from treatment two and one pig from treatment one exhibited hepatocellular damage. The liver sections from the remaining animals were diagnosed as having no visible lesions. 90 ~ I 0~0 TREATMENT I -" -- TREATMENT 2 [3----[3 TREATMENT ?- TREATMENT 4 0 m 6O 5O i i i L L TIME (days) Figure 1. Cumulative mean body weights of pigs that consumed graded levels of aflatoxin. Treatment 1, 20 ppb; treatment 2, 370 ppb; treatment 3, 740 ppb; and treatment 4, 1,480 ppb aflatoxin. == o I I I i I TIME (doys) Figure 2. Cumulative feed efficiency (feed to gain ratio) of pigs that consumed graded levels of aflatoxin. Treatment 1, 20 ppb; treatment 2, 370 ppb; treatment 3,740 ppb; and treatment 4, 1,480 ppb aflatoxin.

4 EFFECTS OF AFLATOXINS ON FINISHING SWINE 1009 TABLE 3. EFFECT OF GRADED LEVELS OF AFLATOXIN ON SERUM PROTEINS IN FINISHING SWINE Blood Dietary aflatoxin level (ppb) a fraction b SE c TSP Albumin d " IgG d IgM d 5.93 e 6.58e, f 6.91 f,g 7.63g.28 aeach treatment contained two replications of four pigs each, average initial weight of 50 and 55 kilograms. bmeans for seven pigs only. CStandard error of the mean, SD/fN. dalbumin, lgg and IgM are calculated as relative percent of total protein. e'f'gmeans with the same or no superscript are not significantly different. (P>.05). Discussion Experimental aflatoxicosis has been induced in swine by feeding diets that contained known concentrations of aflatoxin. Duthie et al (1966) observed no difference in gain or F/G ratio in finishing swine (64 to 91 kg) fed diets containing 410 ppb aflatoxin B1. Duthie et al. (1968) later reported that 690 ppb aflatoxin Bl did not alter performance of finishing swine (54 to 91 kg), but mild histopathological lesions of the liver were detected. Similarly, Hintz et al. (1967) reported that 480 ppb total aflatoxin (450 ppb BI ) did not affect ADG or F/G ratio, but that 765 ppb (615 ppb B1 ) reduced weight gain and 1,005 ppb (810 ppb B1) dramatically reduced ADG and appeared to impair feed efficiency. Monegue et al. (1977a) reported that 400 ppb aflatoxin had a detrimental effect on ADG and feed consumption. In a similar study, Monegue et al (1977b) concluded that 300 ppb did not alter ADG, ADF or F/G ratio in finishing swine (54 kg to market weight). In the present study, ADG and ADF were reduced when 385 ppb total aflatoxin was consumed. Feed efficiency was not affected by the diets that contained 385 and 750 ppb aflatoxin, but pigs that consumed 1,480 ppb aflatoxin required more feed per unit gain than the control pigs. This study agrees with the work of Monegue et al (1977a,b) indicating that the tolerance level of total aflatoxin is less than 400 ppb for finishing pigs. Some of the differences in performance reported above may be explained by different TABLE 4. EFFECT OF GRADED LEVELS OF AFLATOXIN ON THE LIVERS OF FINISHING SWINE Dietary aflatoxin level, (ppb) a Criteria b SE c Pig weight, kg Liver weight, kg Liver weight d 1.31 e 1.55 f 1.58 f 1.77g [04 Visible lesions aeach treatment contained two replications of four pigs each, average initial weight of 50 to 55 kilograms. bmeans for seven pigs only. CStandard error of the mean, SD/4-N. dliver weight expressed as a percent of live body weight. e'f'gmeans with the same or no superscript are not significantly different (P>.05).

5 1010 SOUTHERN AND CLAWSON levels of aflatoxin B2, G1 and G 2. However, this is doubtful since aflatoxin B 1 is the major metabolite present in the swine diets of each of the above mentioned studies and is more than twice as toxic as either aflatoxin B2, G1 or G 2 (Carnaghan et al. 1963). The effects on performance of aflatoxin B2, G1 or G2 would be overshadowed by the effects of aflatoxin B1. Pigs that received the diet containing 750 ppb aflatoxin showed a reduction in ADG during the first 28 days of aflatoxin consumption. In pigs fed the diet containing 385 ppb aflatoxin, this reduction was not evident until 42 days. No difference (P>.10) in feed efficiency was observed between treatments during the initial 28 days of toxin consumption (figure 2). Pigs had not exhibited apparent symptoms of aflatoxicosis during this period, and they all appeared to be as strong and energetic as the controls. Monegue et al. (1977a)suggested that the mold and (or) toxin may have an appetite depressing effect. Murphy et al (1975) fed growing pigs (20 kg starting weight) a diet with the protein and protein-free portion fed separately or combined. The protein portion contained.a high level of aflatoxin. The authors concluded that the reduction in performance was due to a decrease in energy consumption. Average daily feed intake and ADG appear to be more sensitive parameters than feed efficiency in diagnosing aflatoxicosis. This is evident when comparing the results of the ADF, ADG and F/G ratio in table 2. A comparison of figures 1 and 2 illustrates that with time, ADF and ADG were more subject to change than was feed efficiency. The data reported by Hintz et al. (1967) support this conclusion. They reported that feed efficiency was altered in pigs fed 1,005 ppb total aflatoxin but not when 765 ppb aflatoxin was fed; however, both levels caused a reduction in daily gain. Armbrecht et al. (1971) concluded that F/G ratio was more sensitive in diagnosing subclinical aflatoxicosis than was growth. Total serum protein (TSP), albumin and IgG fractions of the gamma globulins were not affected by aflatoxin consumption. The IgM fraction was increased in the serum of pigs that received diets containing 750 and 1,480 ppb aflatoxin when compared to the controls (table 3). These results are not in agreement with the results of Cysewski et al (1968) who reported a decrease in TSP and all serum protein components (alpha, beta and gamma fractions). Annau et al. (1964) observed an increase in the gamma globulins but a decrease in the TSP and serum albumin fraction. Armbrecht et al. (1971) reported similar results with a decrease observed in albumin and an increase in the gamma globulins. The differences that exist between the present results and those of the above workers in TSP and the serum protein fractions may be due to differences in age and weight of animals used or to different levels of aflatoxin consumption. Cysewski et al (1968)determined the clinical features of acute aflatoxicosis in young swine (11.4 to 21.4 kg) by administering high levels of aflatoxin via a gastric fistula; whereas, in the present study, the aflatoxin was ingested over a 5-week period before blood samples were taken. Although differences involving the effects of aflatoxin on serum protein components are present in the literature, one can conclude that aflatoxin consumption increases the gamma globulins, especially the IgM fraction, while TSP and serum albumin are decreased or remain unchanged. The results of the short withdrawal period suggest that pigs that consumed the aflatoxin diets were not so severely affected that they could not resume growth on aflatoxin-free corn. However, liver weights were still enlarged after the withdrawal period and histopathological liver lesions were observed. Liver weights of pigs that remained on the aflatoxin diets were not different from those of pigs switched to control diet during the withdrawal period. It appears from this study and the results of others that approximately 385 ppb total aflatoxin is close to the maximum tolerance level for finishing swine. Depressed growth rate, increased liver weights, abnormal histology and altered serum globulin patterns resulted when diets containing aflatoxin levels of 385 ppb or greater were fed. Literature Cited Annau, E., A. H. Corner, S. E. Magwood and K. Jericho Electrophoretic and chemical studies on sera of swine following the feeding of toxic groundnut meal. Can. J. Comp. Med. 28:264. AOAC Official Methods of Analysis (12th Ed.). Association of Official Analytical Chemists, Washington, DC. Armbrecht, B. H., H. G. Wiseman, W. T. Shalkop and J. N. Geleta Swine aflatoxicosis. I. An assessment of growth efficiency and other responses in growing pigs fed aflatoxin. Environ. Physiol. 1:198.

6 EFFECTS OF AFLATOXINS ON FINISHING SWINE 1011 Barr, A. J, and J. H. Goodnight A user's guide to the statistical ~nalysis system (1st Ed.). North Carolina State Univ., Raleigh. Carnaghan, R. B. A., R. D. Harttey and J. O'Kelly Toxicity and fluorescence properties of the aflatoxins. Nature 200:1101. Cysewski, S. J., A. C. Pier, G. W. Engstrom, J. L. Richard, R. W. Dougherty and J. R. Thurston Clinical pathologic features of acute aflatoxicosis of swine. Amer. J. Vet. res. 29:1577. Davis, B. J Disc electrophoresis. II. Method and application to human serum proteins. Ann. NY Aead. Sei. 121:404. Duthie, I. F.~ M. C. Lancaster, J. Taylor~ D. C. Thomas, C. A. Shacklady, P. H. Altfield and F. Lewis Toxic groundnut meal in feed for pigs. 1_ A trial made at two laboratories with pigs from about 40 to 200 lbs live weight fed a restricted scale. Vet. Rec. 79:621. Duthie, L F., M. C. Lancaster, J. Taylor, E. B. Lomas and H M. Clarkson Toxic groundnut meal in feeds for pigs. I. The effect of consuming groundnut meal during part of the growing period or during the finishing period. Vet. Rec. 82:427. Hintz, H. F., A. N. Booth, A. F. Cucullu, H. K. Gardner and H. Heitman, Jr Aflato toxicity in swine. Proc. Soc. Exp. Biol. Med. 124: 266. Lowry, O. H., N. J. Rosebrough, A. L. Farr and R. J. Randall Protein measurement with the folin phenol reagent. J. Biol. Chem. 193 : 265. Monegue, H. J., G. E, Combs, G. T. Edds and H. D. Wallace. 1977a. A preliminary study of the effects of aflatoxin on young swine. Florida Agr. Exp. Sta. Gainesville. Research Rep. AL Monegue, H. J., G. E. Combs, G. T. Edds and H. D. Wallace. 1977h. The effects of various levels of aflatoxins on young swine. Florida Agr. Exp. Sta., Gainesville, Research Rep. AL Murthy, T. R. K., M. Lemmali, Y. Henry and C. Frayssinet Aflatoxin residues in tissues of growing swine: Effect of separate and mixed feeding of protein and protein-free portions of the diet. J. Anita. Sci. 41:1339. Smith, R. B. Jr., M. J. Griffin and P. B. Hamilton Survey of aflatoxicosis in farm animals. Appl. Environm. Microbiol. 31:385.