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Effects of Feeding Fusarium moniliforme Culture Material, Containing Known Levels of Fumonisin B1, on the Young Broiler Chick1
Effects of Feeding Fusarium moniliforme Culture Material, Containing Known Levels of Fumonisin B1; on the Young Broiler Chick1 T. S. WEIBKING,2 D. R. LEDOUX,2'3 A. J. BERMUDEZ,4 J. R. TURK,4 and G. E. ROTTINGHAUS4 Department of Animal Sciences, and Veterinary Medical Diagnostic Laboratory, College of Veterinary Medicine, University of Missouri, Columbia, Missouri 65211 E. WANG and A. H. MERRILL, JR.
ABSTRACT The effects of feeding Fusarium moniliforme culture material, containing known concentrations of fumonisin Bi (FBi), were studied in broiler chicks. Day-old chicks were allotted randomly to dietary treatments containing 0,1.02, 2.04, 3.06,4.08,5.10,6.12, and 7.14% fumonisin culture material (FCM). These levels of FCM supplied 0,75,150,225,300,375,450, and 525 mg of FBi /kg of feed. Each dietary treatment was fed to four pen replicates of six birds each for 21 days. Chicks fed FCM that supplied 450 and 525 mg FBi/kg diet had lower (P < .05) feed intakes and BW gains; increased (P < .05) liver and kidney weights; and increased (P < .05) mean cell hemoglobin, and mean cell hemoglobin concentrations. Compared with controls, chicks fed FCM had increased (P < .05) free sphinganine levels and sphinganine:sphingosine ratios. Treatmentassociated histological lesions were only observed in the liver of chicks fed diets containing FCM that supplied 225 mg FBi/kg or higher. Diets containing FCM that supplied levels as low as 75 mg FBi/kg affected the physiology of chicks by increasing free sphinganine levels and sphinganine:sphingosine ratios. Because inhibition of sphingolipid biosynthesis has been hypothesized as the mechanism of action of FB^ this suggests that diets containing 75 mg FBi/kg FCM may be toxic to young broiler chicks. (Key words: fumonisin Bi, broilers, Fusarium moniliforme, sphinganine, sphingosine) 1993 Poultry Science 72:456-466
reduced gains, high mortality, and development of lesions in the oral cavity Poultry rations with high levels of (Wyatt, 1987; Englehardt et al., 1989; Wu et Fusarium contamination have been as- ah, 1991). In many instances, the predomisociated with poor performance, diarrhea, nant species found was Fusarium moniliforme. There are also several reports of FMSfln'Mm-contaminated grains causing mortality in wild birds (Hagler and ParkReceived for publication July 20, 1992. hurst, 1990; Nelson et al, 1990). Accepted for publication November 24, 1992. iContribution Number 11721 of the Missouri Recently, a new group of water-soluble Agricultural Experiment Station. toxins produced by F. moniliforme has been department of Animal Sciences. characterized. These toxins, which may be 3 To whom correspondence should be addressed: S142 Animal Sciences Center, Columbia, MO 65211. responsible for the toxicological effects of 4 F. moniliforme, are collectively called Veterinary Medical Diagnostic Laboratory. INTRODUCTION
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Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322
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EFFECTS OF FUMONISIN ON BROILERS
MATERIALS AND METHODS
Sphlngoslne OH
OH
OH
OH
OH
Fumonisin B, (backbone)
FIGURE 1. Structures of sphinganine, sphingosine, fumonisin Bj backbone, and fumonisin Bj.
Experimental Design and Birds
One hundred and ninety-two day-old during autoclaving. Whole shelled corn female Arbor Acres x Peterson broiler (100 g) and 100 mL of distilled water were chicks were allotted randomly to pens in a added to each jar, autoclaved for 30 min at stainless steel chick battery and allowed to 121 C and a pressure of 1.4 kg/cm 2 , and consume feed and water ad libitum. The allowed to cool to room temperature. experimental design consisted of eight Fusarium moniliforme M-13255 was added to dietary treatments with four pen replicates sterilized distilled water and 2 mL of the of six birds allotted randomly to each suspension was added to the autoclaved dietary treatment. The day-old chicks were jars. A sterilized glass fiber filter was placed fed experimental diets from hatching to 21 inside the screw-on lid and the autoclave days of age. Chicks were monitored daily tape was removed from the exterior side of for signs of morbidity and mortality. the lid. The jars were shaken and lids loosened to allow for additional respiration. After 24 h of incubation at 27 C in the dark, Fumonisin Production and Analysis the jars were shaken again to insure Cultures were grown in .946-L canning complete dispersal of the fungal mycelium. jars. The lids were modified to allow more The jars were incubated in the dark for a air exchange by drilling three holes (.635- total of 5 wk at 27 C. cm) and covering them with autoclave tape A 400-mL mixture of acetonexhloroform (75:25) was added directly to each 5-wk-old culture and allowed to stand 5 Fusarium Research Center, Pennsylvania State overnight. The contents of each jar were placed in a blender for 30 s and vacuum University, University Park, PA 16802.
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fumonisins and include fumonisin Ai, A2, Bi, B2/ B3, and B4 (Gelderblom et al, 1992). Fumonisin Bj (FBi; Figure 1), the major compound, has been shown to be the causative agent in equine leukoencephalomalacia (Marasas et al, 1988) and porcine pulmonary edema (Harrison el al, 1990). Fumonisin Bi is hepatotoxic in rats (Voss et al, 1990) and has been shown to cause morphological and functional changes in chicken macrophages in vitro, which implies an immunosuppressive effect (Qureshi and Hagler, 1992). Initial studies with broilers indicated that high concentrations of FBi present in F. moniliforme cultures caused poor performance and increased organ weights, diarrhea, multifocal hepatic necrosis, biliary hyperplasia, and rickets (Brown et al, 1992; Ledoux et al, 1992). The objectives of the present study were to investigate further the toxic effects of FBi, present in F. moniliforme cultures, in young broilers and to determine the minimum dietary concentration necessary to cause deleterious effects.
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WEIBKING ET AL.
filtered through a large buchner funnel containing fast filter paper.6 The solid culture residue was reextracted with acetonexhloroform (75:25), refiltered, and allowed to air dry overnight in the hood. The culture material was placed in a forceda ir oven at 40 C for 48 h and ground to a fine powder in a mill.7 The culture material was analyzed for FBlr FB2, and FB3 by HPLC by the procedure of Wilson et al. (1990). Diet Preparation
Sample Collection
At the end of Weeks 1, 2, and 3 of the experiment, chicks were weighed individually and feed consumption was determined for each pen. On Day 21, blood samples were collected via cardiac puncture for serum biochemistry and sphingolipid analysis and for hematologic determinations. Serum biochemical values were determined using an autoanalyzer.8 Hemoglobin was
6
Whatman International Ltd., Maidstone, England. TFitzmill Model D Comminuting machine, Fitzpatrick Co., Elmhurst, IL 60126. 8 Kodak Ektachem Analyzer, Eastman Kodak Co., Rochester, NY 14650. 9 Sigma Diagnostics, Sigma Chemical Co., St. Louis, MO 63178-9916. '"Coulter Counter Model ZB1, Coulter Electronics, Hialeah, FL 33010.
Histopathology
Post-mortem examinations were performed on six chicks from each treatment group at 3 wk of age. Chicks were anesthetized with CO2, euthanatized by cervical dislocation, and samples of proximal tibiotarsal bone, pectoral muscle, adductor muscle, ventriculus, proventriculus, lung, spleen, thymus, bursa of Fabricius, heart, duodenum, pancreas, jejunum, ileum, cecum, kidney, and liver were fixed in 10% neutral-buffered formalin. Fixed tissues were trimmed, embedded in paraffin, sectioned at 4 /«m, and stained with hematoxylin and eosin stain. All tissues from the control and from groups receiving FCM that supplied 450 and 525 mg FBi/kg were examined microscopically. Liver sections from all experimental groups were examined microscopically, liver histopathology was graded in a blind fashion, and lesion scores were reported. Statistical Analysis
Data were analyzed by the General Linear Model procedure of SAS® software (SAS Institute, 1985) as a completely randomized design. Mean differences were determined using Fisher's least significant difference test. Absolute organ weights were adjusted for final body weight by covariance analysis (Shirley, 1977). Statistical significance was accepted at P < .05. RESULTS
The effects of dietary FCM on chick performance are presented in Table 1.
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Dietary treatments were prepared by substituting ground F. moniliforme M-1325 culture material for ground corn in a typical corn-soybean meal basal diet. Fumonisin culture material (FCM) contained 7,800 mg FBj/kg by analysis and made up 0, 1.02, 2.04, 3.06, 4.08, 5.10, 6.12, and 7.14% of the respective diets and supplied 0,75,150,225, 300, 375, 450, and 525 mg FBx/kg diet, respectively. Diets were formulated to be isocaloric and isonitrogenous and either met or exceeded the nutrient requirements of broiler chicks as recommended by the National Research Council (1984). Diets and culture material were screened by the method of Rottinghaus et al. (1982) and found to be free of the following mycotoxins: anatoxin, citrinin, sterigmatocystin, zearalenone, ochratoxin A, T-2 toxin, diacetoxyscirpenol, and vomitoxin.
measured as cyanmethemoglobin.9 Red blood cell counts, mean cell volume, and hematocrits were determined with a counter10 using instrument settings described by Steel et al. (1977). Mean cell hemoglobin (MCH) and mean cell hemoglobin concentration (MCHC) were calculated. Serum sphingolipids, free sphinganine (SA), and free sphingosine (SO), were determined by HPLC using the procedures described by Merrill et al. (1988). Following blood sampling, 12 chicks per treatment were euthanatized by asphyxiation with CO2, and selected organs were excised and weighed.
459
EFFECTS OF FUMONISIN ON BROILERS TABLE 1. Effects of Fusarium moniliforme culture material, containing fumonisin, on performance of broiler chicks Culture material
Days
1 to 7
8 to 14
15 to 21
1 to 21
(r-V
(%) 0 1.02 2.04 3.06 4.08 5.10 6.12 7.14 SE
(0)2 (75) (150) (225) (300) (375) (450) (525)
109 105 108 108 107 108 99 99 3
290» 266b«i 292a 280 abcd 283 abc 287ab 261 cd
99a 100' 99a 97a 95ab 97a 88b 88b 3
232 219 231 220 226 225 212 209 6
260=*
8
461 454 460 456 459 459 445 441 6
860a 838 ab 859a 844 ab 849 a 854a 809bc 796c 13
B o d y w e i g h t g a i n (g) 1
(0) (75) (150) (225) (300) (375) (450) (525)
338 328 330 321 334 335 319 314 9
669 a 646 al * 659 a 636 al * 654 a 657 a 6181* 610c 14
Feed c o n v e r s i o n (feed:gain) (g:g) a
0 1.02 2.04 3.06 4.08 5.10 6.12 7.14 SE
(0) (75) (150) (225) (300) (375) (450) (525)
1.10' 1.05b 1.09»b 1.12' 1.12' 1.11» 1.13a l.W .02
1.25 1.22 1.27 1.27 1.25 1.27 1.23 1.24
.03
1.37 1.39 1.39 1.42 1.38 1.37 1.40 1.41 .04
1.29 1.30 1.30 1.33 1.30 1.30 1.31 1.31 .02
a_d
Values for variables within columns with no common superscripts differ significantly (P < .05). !Data are means of four replicate pens of six chicks each. 2 Dietary levels (milligrams per kilogram) of fumonisin Bj.
Compared with controls, chicks fed FCM that supplied 450 and 525 mg FBi/kg diet had reduced feed intakes at Week 2, and reduced BW gains at Week 1. Over the combined 3-wk period, chicks fed FCM that supplied 450 and 525 mg FB^/kg had reduced feed intakes and lower BW gains compared with controls. With the exception of a modest improvement in feed conversion in chicks fed FCM that supplied 75 mg FBi/kg at Week 1, feed conversion was not affected by dietary treatment. Compared with controls, increased liver weights were observed in chicks fed FCM that supplied 450 and 525 mg FBj/kg, and increased kidney weights were observed
in chicks fed FCM that supplied 375 to 525 mg FBi/kg (Table 2). Chicks fed FCM that supplied 525 mg FBi/kg had heavier proventriculus weights compared with control chicks (Table 2). Bursa of Fabricius, gizzard, and heart weights were not influenced by dietary FCM (Table 2). Compared with controls, MCH and MCHC were higher in chicks fed FCM that supplied 450 and 525 mg FBi/kg diet (Table 3). Red blood cell counts, hemoglobin, hematocrits, and mean cell volume were not influenced by dietary FCM (Table 3). With the exception of modest declines in serum cholesterol levels in chicks fed FCM that supplied 75 and 300 mg FBi/kg, serum chemistry values were
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0 1.02 2.04 3.06 4.08 5.10 6.12 7.14 SE
460
WEIBKING ET AL. TABLE 2. Effects of Fusarium moniliforme culture material, containing fumonisin, on absolute organ weights of broiler chicks1'2
Culture material
Liver
Kidney
Proventriculus
17.41** 17.21= 16.80= 16.98= 17.01= 19.03* 19.20" 19.42*
6.93=* 6.81* 7.38ocde 7.37cde 7.64t>«! 8.07* 7.86ab= 8.65* .28
4.41 b 4.55b 4.39b 4.36b 4.66b 4.85* 4.54b 5.34*
(%) 0 1.02 2.04 3.06 4.08 5.10 6.12 7.14 SE
Bursa of Fabricius
Gizzard
Heart
1.55 1.78 1.46 1.97 1.62 1.65 1.55 1.75 .13
16.01 17.39 17.79 17.49 17.47 18.10 16.95 17.78 .57
4.52 4.28 4.36 4.40 4.18 4.46 4.29 4.92 .27
(0)3 (75) (150) (225) (300) (375) (450) (525)
.59
\g)
.19
a_e
not affected by FCM (Table 4). Compared with controls, chicks fed all levels of FCM had increased free SA levels and SA:SO ratios (Figure 2). Free SO levels were not affected by dietary treatments. No lesions were seen in skeletal muscle, spleen, thymus, heart, kidney, proventriculus, lung, bursa of Fabricius, and duodenum, pancreas, jejunum, ileum, cecum, or proximal tibiotarsus of the control or treatment groups. Control and treatment groups had
incidental histologic features in the ventriculus. Mild multifocal areas of heterophil infiltration of the ventricular mucosa were associated with erosions in the koilin lining. Lesions associated with FCM treatment were observed only in the liver. A periportal granulocytic cell proliferation (Figure 3) was evident in control and treatment groups and this lesion progressed from mild to severe with
TABLE 3. Effects of Fusarium moniliforme culture material, containing fumonisin, on hematology of broiler chicks1'2 Culture material
HCT 3
(%) 0 1.02 2.04 3.06 4.08 5.10 6.12 7.14 SE
RBC
(0)3 (75) (150) (225) (300) (375) (450) (525)
a_c
(xlOVmm ) 2.59 2.66 2.50 2.60 2.49 2.47 2.44 2.48 .06
(%) 34.7 35.0 33.4 34.4 33.1 33.0 32.5 33.3 .8
HB (g/dL) 8.4 9.2 8.2 8.2 8.5 8.3 8.8 9.0 .3
MCV 3
0*m ) 127 125 127 126 126 127 126 127 1
MCH
MCHC
(Pg) 32.3b= 34.8* 32.3 bc 31.4= 34.0 abc 33.3 abc 36.0' 36.1 a 1.0
(g/dL) 24.1= 26.3 ab 24.1= 23.7= 25.5 abc 24.8b= 27.1 a 26.7 ab
Values within columns with no common superscripts differ significantly (P < .05). *Data are means of four replicates of three chicks each. 2 RBC = red blood cells; HCT = hematocrit; HB = hemoglobin; MCV = mean cell volume; MCH = mean cell hemoglobin; MCHC = mean cell hemoglobin concentration. 3 Dietary levels (milligrams per kilogram) of fumonisin B^.
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Values within columns with no common superscripts differ significantly (P < .05). 1 Absolute organ weights adjusted for final body weight by analysis of covariance. 2 Data are means of four replicates of three chicks each. 3 Dietary levels (milligrams per kilogram) of fumonisin Bj.
461
EFFECTS OF FUMONISIN ON BROILERS TABLE 4. Effects of Fusarium moniliforme culture material, containing fumonisin, on serum chemistry of broiler chicks1 Culture material
(%) 0 1.02 2.04 3.06 4.08 5.10 6.12 7.14 SE
(0)4 (75) (150) (225) (300) (375) (450) (525)
a_c
Glucose (mg/dL) 289 269 273 286 264 280 277 269 9
Total protein 3.45 3.10 3.26 3.22 3.21 3.13 3.35 3.21 .12
Albumin
- (g/dL) 1.39 1.26 1.35 1.31 1.33 1.29 1.35 1.33 .05
-
Cholesterol (mg/dL) 134^ 107< 120 abc H7abc H4bc 128ab 135* 126"'' 7
GGT2
AST3
- (IU/L) 31.5 30.3 33.9 29.1 35.0 32.5 34.2 35.9 1.8
Values within columns with no common superscripts differ significantly (P < Data are means of four replicates of two chicks each. 2 GGT = 7 glutamyl transferase. 3 AST = aspartate aminotransferase. 4 Dietary levels (milligrams per kilogram) of fumonisin Bj.
185 201 191 170 211 240 212 259 22
.05).
:
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increasing doses of FCM in the feed (Table days and resulted in poor feed conversion. 5). The cytoplasm of most of these No diarrhea was observed in the present granulocytic cells contained large spheri- study and as a result, feed conversion was cal eosinophilic granules and a large not affected by dietary treatment. In round, eccentrically located nucleus. Iso- earlier studies, FBi concentrations in FCM lated mature heterophils were also present averaged 2,000 mg/kg or less, and in in these periportal infiltrates as well as order to achieve the required dietary rare foci of erythropoiesis. These peripor- concentrations it was necessary to add tal granulocytic infiltrates were inter- significant quantities (>10%) of FCM to preted to be the extramedullary the diets. Diarrhea appears to occur only hematopoiesis. Isolated foci of hepatic when the level of FCM exceeds 10% of the necrosis with a mild heterophil and mac- diet. In the present study, the highest level rophage infiltration (Figure 3) were ob- of FCM incorporated in diets was 7.14%. served in chicks fed FCM that supplied Two possible mechanisms by which FCM 225 mg FBi/kg or greater. Moderate producing low concentrations of FBi cause diffuse hepatocellular hyperplasia was diarrhea are proposed. An additional toxic present in liver sections of chicks fed FCM metabolite may be present in F. that supplied 225 mg/kg FBi and greater. moniliforme cultures that produce low Mild biliary hyperplasia was present in concentrations of FBj. This hypothesis is isolated liver sections of chicks fed FCM supported by a recent report (Norred et that supplied 225 mg FBi/kg or greater in al, 1991) indicating the presence of other cytotoxic water-soluble metabolites in F. the ration. moniliforme cultures. A second possibility is that an unknown toxic metabolite is DISCUSSION present in all F. moniliforme cultures at a Depression in BW gains observed in low concentration, and a diarrheathis study (7 to 9%) was less than that producing dose of this metabolite is not observed in previous studies with broilers administered until greater than 10% FCM (Brown et al, 1992: 19%; Ledoux et al, is included in the ration. The increase in liver weight observed in 1992: 13%) and turkeys (Weibking et al, 1992: 19%) fed FCM that supplied 100 to this study was observed in previous 400 mg FBj/kg diet. In previous studies, a studies with broilers fed FCM that supsevere diarrhea was present after 8 to 10 plied 100 to 400 mg FBi/kg (Brown et al,
WEIBKING ET AL.
462
•
# •
Hi 150
225
300
375
450
525
600
B 150
o
a-,
CO DC
7 -
0)
c
Si
300
375
450
525
6-
o O) c x: Q. 01
,5.
0) C
.3-
c CO
225
FIGURE 3. A) Severe periportal extramedullary hematopoiesis (arrows) in liver of chick fed 525 mg fumonisin Bj/kg. Original magnification = l,000x (bar = 15 /zm). B) Hepatic necrosis (N) with mild heterophil and macrophage infiltrate in liver of chick fed 375 mg fumonisin Bj/kg. Original magnification = 200x (bar = 50 urn).
4-
.2-
O)
c .1 •
c
a.
w -75
0
75
150
225
300
Fumonisin B1
375
450
525
600
(mg/kg)
FIGURE 2. Effects of fumonisin Bj on sphinganine (A), sphingosine (B), and sphinganine: sphingosine ratio (C). Compared with controls, chicks fed diets containing fumonisin Bj had increased (P < .05) free sphinganine levels and sphinganine:sphingosine ratios.
1992; Ledoux et ah, 1992) and turkeys fed FCM that supplied 100 to 200 mg FB a /kg (Weibking et al., 1992). In contrast to broilers and turkeys, dietary FB! from FCM has been shown to decrease absolute liver weights in rats fed 93 to 139 mg FBi/ kg (Voss et al, 1990). Increases in proventriculus weights in response to FCM have also been observed previously in broilers fed FCM that supplied 100 to 400 mg FBi/ kg (Ledoux et al, 1992). Increased kidney weights observed in the present study were not seen previously in broilers fed FCM that supplied 100 to 400 mg F B ^ k g (Ledoux et al, 1992) but were seen in turkeys fed FCM that supplied 100 to 200
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75
463
EFFECTS OF FUMONISIN ON BROILERS
TABLE 5. Summary of hepatic lesions in chicks fed diets containing Fusarium moniliforme culture material with known levels of fumonisin Bj 1
Culture material
vjo esions
Periportal granulocy tic cell proliferation Severe Mild Moderate
Hepatic necrosis
Hepato-cellular hyperplasia
1/6 2/6 4/6 2/6 4/6
1/6
(%) (0)2 (75) (150) (225) (300) (375) (450) (525)
0 1.02 2.04 3.06 4.08 5.10 6.12 7.14
2/6
4/6 5/5 6/6 5/6 3/6 2/6 4/6
1/6 3/6 4/6 2/6 5/6
1/6
3/6 1/6 3/6
1
Number with trait/total. Dietary levels (milligrams per kilogram) of fumonisin Bj.
2
The failure of FCM to affect serum aspartate aminotransferase (AST) activity in the present study is surprising because increased serum AST activity has previously been observed in chicks (Ledoux et al, 1992), turkeys (Weibking et al, 1992), rats (Voss et al, 1990), and pigs (Osweiler et al, 1992) fed FBi. An increase in serum AST values is indicative of altered hepatocyte membrane integrity with the leakage of this enzyme or hepatic necrosis (Duncan and Prasse, 1986). A possible explanation for the difference between the present study and those reported previously can be found in Table 5. There was a lack of uniformity in the incidence of hepatic necrosis in chicks fed FCM that supplied 225, 300, 375, 450, and 525 mg FBi/kg. This resulted in a large standard error for mean AST values, and as a result no statistical differences were observed. It is important to note however, that individual elevated AST values are true values that correspond to individual chicks that had hepatic necrosis. Wang et al (1991), noting the remarkable structural similarity of the fumonisin backbone to SO and SA (Figure 1), hypothesized that disruption of SO metabolism might be a target of the fumonisins. In studies aimed at proving this hypothesis, Wang et al (1991) found that FBi inhibited de novo sphingolipid biosynthesis and proposed that this inhibition may be the initial molecular target of these toxins (Norred et al, 1992). Fumonisin Bj inhibition of sphingolipid biosynthesis resulted
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mg FBi/kg (Weibking et al, 1992). Other mycotoxins produced by the genus Fusarium have been reported to cause various hematological effects in broilers. Deoxynivalenol has been reported to decrease red blood cell counts and hematocrits (Huff et al, 1986), MCH (Kubena et al, 1988), and mean cell volume (Kubena et al, 1989). The T-2 toxin has been reported to cause decreased hemoglobin, mean cell volume, and MCHC (Kubena et al, 1989). In the current study, the hematological changes observed were an increase in MCH and MCHC in chicks fed FCM that supplied 450 and 525 mg FBi/kg. These mild changes are interpreted to be the result of increased red blood cell hemolysis with increasing levels of FCM in the ration. Hemolysis will cause a false elevation in MCH and MCHC because free hemoglobin will be attributed to the remaining nonhemolyzed red blood cells in the sample (Jain, 1986). Hemolysis will also result in a false decrease in red blood cell counts based on Coulter counter analysis (Jain, 1986). Both of these changes will be reflected in increased MCH and MCHC indexes. The interpretation of these results is that increased FCM in the ration may be associated with increased red blood cell fragility. It should be noted that all hematological parameters of all treatment groups were within normal ranges found in chicks Qain, 1986). Increases in MCH and MCHC were not observed in turkeys fed FCM that supplied 100 or 200 mg FBi/kg (Weibking et al, 1992).
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WEIBKING ET AL.
Results indicate that diets containing FCM that supplies 225 m g / k g or higher FBi cause hepatic lesions in young broilers. However, diets containing FCM that supplied levels as low as 75 mg FBi/kg affected the physiology of chicks by causing an increase in SA levels and SA:SO ratios. Because inhibition of sphingolipid biosynthesis has been hypothesized as the mechanism of action of FBi, this suggests that FBi toxicity may be of practical concern in broilers, as naturally occurring levels of FBi as high as 189 m g / k g have been reported in corn (Ross et al, 1991), and most broiler rations would contain a minimum of 50% corn (or 94 m g FBi/kg). The possibility that there may be other toxic metabolites in these F. moniliforme cultures that may be responsible for diarr h e a i m p l i e s t h a t F. moniliforme-
contaminated commodities may also be of concern to the broiler industry. Also of concern are recent reports indicating that in cell culture, FBi caused morphological and functional changes in chicken macrophages (Qureshi and Hagler, 1992) and was cytotoxic to turkey lymphocytes (Dombrink-Kurtzman et al, 1992). If FBi is shown to be immunosuppressive in chicks (in vivo), then levels required to compromise the immune system may well be lower than levels required to produce overt signs of toxicity. Finally, a recent report suggests that FBi may not be the only fumonisin responsible for toxicological effects. Gelderblom et al. (1992) reported that FB2 and FB3 are as toxic as FBi. If so, then the total fumonisin B levels should be used to evaluate the toxic effects of fumonisins. In the present study, total dietary fumonisin B levels (FBi + FB2 + FB3) were 0, 89,190, 283, 389, 481, 592, and 681 m g / k g , respectively. ACKNOWLEDGMENTS This study was supported in part by the National Institutes of Health Institutional Biomedical Research S u p p o r t Grant RR07053; by USDA Animal Health Formula Funding: Development of Large Scale Preparative and Analytical Methodology for the Mycotoxin, Fumonisin Bi, produced by Fusarium moniliforme; and by USDA Grant 91-37204-6684: Molecular mechanisms of animal diseases caused by fumonisin. The authors thank Paul E. Nelson, Pennsylvania State University, University Park, PA 16802 for supplying the F. moniliforme cultures; and Frank Ross and Larry Rice, National Veterinary Services Laboratories, Animal Plant Health Inspection Service, USDA, Ames, IA 50011, for confirmation and analytical assistance. REFERENCES Brown. T. P., G. E. Rottinghaus, and M. E. Williams, 1992. Fumonisin mycotoxicosis in broilers: performance and pathology. Avian Dis. 36:450-454. Dombrink-Kurtzman, G. A., G. A. Bennett, and J. L. Richard, 1992. Avian lymphocytes as an in vitro model to predict fumonisin cytotoxicity. Fed. Am. Soc. Exp. Biol. J. 6(5):A2007. Duncan, J. R., and K. W. Prasse, 1986. Veterinary
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in an accumulation of SA and an increase in the SA:SO ratio in liver hepatocytes (Wang et al, 1991), cultured renal cells (Yoo et al, 1992), and blood serum of ponies (Wang et al, 1992). Similar increases in SA levels and SA:SO ratios were observed in the present study at the lowest level of FCM that supplied 75 mg FBi/kg (Figure 2). Wang et al. (1992) hypothesized that the depletion of complex sphingolipids (resulting from FBi inhibition of biosynthesis) and accumulation of SA may well be the cause of pathology observed in FBi toxicosis, because these sphingolipids are involved in regulation of cell surface receptors, ion pumps, and other systems vital for cell function and survival. The liver lesions observed in these FBiexposed broiler chicks are similar to those reported in FBi-exposed rats, which also developed hepatocellular hyperplasia, biliary hyperplasia, and hepatic necrosis (Voss et al, 1990). The lesions observed in horses, swine, sheep, a n d b a b o o n s [encephalomalacia, pulmonary edema, acute nephrosis, and acute congestive heart failure, respectively; (Kriek et al., 1981)] were not observed in the chicks in this trial. Increased EMH was an unexpected response to increasing levels of dietary FCM. Hepatocellular hyperplasia is a likely mechanism for the increased liver weights observed in this study (Table 3).
EFFECTS OF FUMONISIN ON BROILERS
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Laboratory Medicine: Clinical Pathology. 2nd Norred, W. P., E. Wang, H. Yoo, R. T. Riley, and A. ed. Iowa State University Press, Ames, IA. H. Merrill, Jr., 1992. In vitro toxicology of Engelhardt, J. A., W. W. Carlton, and J. F. Tuite, 1989. fumonisins and the mechanistic implications. Toxicity of Fusarium moniliforme var. subglutiMycopathologia 117:73-78. nans for chicks, ducklings, and turkey poults. Osweiler, G. D., P. F. Ross, T. M. Wilson, P. E. Avian Dis. 33:357-360. Nelson, S. T. Witte, T. L. Carson, L. G. Rice, and Gelderblom, W.C.A., W.F.O. Marasas, R. Vleggar, P. H. A. Nelson, 1992. Characterization of an G. Thiel, and M. E. Cawood, 1992. Fumonisins: epizootic of pulmonary edema in swine asIsolation, chemical characterization and biologisociated with fumonisin in corn screenings. J. cal effects. Mycopathologia 117:11-16. Vet. Diagn. Invest. 4:53-59. Harrison, L. R., B. M. Colvin, J. T. Greene, L. E. Qureshi, M. A., and W. M. Hagler, 1992. Effect of Newman, and R. C. Cole, Jr., 1990. Pulmonary fumonisin-Bj exposure on chicken macrophage edema and hydrothorax in swine produced by function in vitro. Poultry Sci. 71:104-112. fumonisin Bj, a toxic metabolite of Fusarium Ross, P. F., L. G. Rice, R. D. Plattner, G. D. Osweiler, moniliforme. J. Vet. Diagn. Invest. 2:217-221. T. M. Wilson, D. L. Owens, H. A. Nelson, and J. Hagler, W. M., and C. R. Parkhurst, 1990. Probable L. Richard, 1991. Concentrations of fumonisin fusariotoxicosis in domestic waterfowl in North Bj in feeds associated with animal health Carolina. Poultry Sci. 69(Suppl. l):170.(Abstr.) problems. Mycopathologia 114:129-135. Huff, W. F., L. F. Kubena, R. B. Harvey, W. M. Rottinghaus, G. E., B. Olsen, and G. D. Osweiler, Hagler, Jr., S. P. Swanson, T. D. Phillips, and C. 1982. Rapid screening method for aflatoxin Bl, R. Creger, 1986. Individual and combined zearalenone, ochratoxin A, T-2 toxin, diacetoxeffects of aflatoxin and deoxynivalenol (DON, yscirpenol and vomitoxin. Pages 477-484 in: vomitoxin) in broiler chicks. Poultry Sci. 65: Proceedings of the 25th Annual American 1291-1298. Association of Veterinary Laboratory DiagnostiJain, N. C, 1986. Schalm's Veterinary Hematology. cians, Nashville, TN. 4th ed. Lea and Febiger, Philadelphia, PA. SAS Institute, 1985. SAS® User's Guide: Statistics. Kriek, N.P.J., T. S. Kellerman, and W.F.O. Marasas, Version 6 Edition. SAS Institute Inc., Cary, NC. 1981. A comparative study of the toxicity of Shirley, E., 1977. The analysis of organ weight data. Fusarium verticillioides (= F. moniliforme) to Toxicology 8:13-22. horses, primates, pigs, sheep, and rats. OnderSteel, E. G., H. D. Petersen, A. Blanks, and H. E. stepoort J. Vet. Res. 48:129-131. Smalley, 1977. The application of an electronic Kubena, L. F., R. B. Harvey, W. E. Huff, D. E. Corrier, particle counter with a mean cell volume T. D. Phillips, and G. E. Rottinghaus, 1989. computer and an hematocrit accessory to avian Influence of ochratoxin A and T-2 toxin singly hematology. Poultry Sci. 56:839-842. and in combination on broiler chicks. Poultry Voss, K. A., R. D. Plattner, C. W. Bacon, and W. P. Sci. 68:867-872. Norred, 1990. Comparative studies of Kubena, L. F., W. E. Huff, R. B. Harvey, D. E. Corrier, hepatotoxicity and fumonisin Bj and B2 content T. D. Phillips, and C. R. Creger, 1988. Influence of water and chloroform/methanol extracts of of ochratoxin A and deoxynivalenol on growing Fusarium moniliforme strain MRC 826 culture broiler chicks. Poultry Sci. 67:253-260. material. Mycopathologia 112:81-92. Ledoux, D. R., T. P. Brown, T. S. Weibking, and G. E. Rottinghaus, 1992. Fumonisin toxicity in broiler Wang, E., W. P. Norred, C. W. Bacon, R. T. Riley, and A. H. Merrill, Jr., 1991. Inhibition of sphinchicks. J. Vet. Diagn. Invest. 4:330-333. golipid biosynthesis by fumonisins: Implications Marasas, W.F.O., T. S. Kellerman, W.C.A. Gelderfor diseases associated with Fusarium blom, J.A.W. Coetzer, P. G. Thiel, and J. J. Van moniliforme. J. Biol. Chem. 266:14486-14490. Der Lugt, 1988. Leukoencephalomalacia in a Wang, E., P. F. Ross, T. M. Wilson, R. T. Riley, and A. horse induced by fumonisin Bj isolated from H. Merrill, Jr., 1992. Increases in serum sphingoFusarium moniliforme. Onderstepoort J. Vet. Res. sine and sphinganine and decreases in complex 55:197-203. sphingolipids in ponies given feed containing Merrill, A. H., Jr., E. Wang, R. E. Mullins, W.C.L. fumonisins, mycotoxins produced by Fusarium Jamison, S. Nimkar, and D. C. Liotta, 1988. moniliforme. J. Nutr. 122:1706-1716. Quantitation of free sphingosine in liver by high-performance liquid chromatography. Anal. Weibking, T. S., D. R. Ledoux, T. P. Brown, and G. E. Rottinghaus, 1992. Fumonisin toxicity in turBiochem. 171:373-381. keys. J. Vet. Diagn. Invest, (in press). National Research Council, 1984. Nutrient Requirements of Broilers. Pages 13-14 in: Nutrient Wilson, T. M., P. F. Ross, L. G. Rice, G. D. Osweiler, H. A. Nelson, D. L. Owens, R. D. Plattner, C. Requirements of Poultry. 8th rev. ed. National Reggiardo, T. H. Noon, and J. W. Pickrell, 1990. Academy Press, Washington, DC. Fumonisin Bj levels associated with an Nelson, P. E., R. J. Cole, T. A. Toussoun, J. W. epizootic of equine leukoencephalomalacia. J. Dorner, and R. M. Windingstad, 1990. Fusarium Vet. Diagn. Invest. 2:213-216. species recovered from waste peanuts associated with sandhill crane mortality. Mycolo- Wu, W., M. E. Cook, and E. B. Smalley, 1991. gia 82(5):562-565. Decreased immune response and increased incidence of tibial dyschondroplasia caused by Norred, W. P., C. W. Bacon, R. D. Plattner, and R. F. fusaria grown on sterile corn. Poultry Sci. 70: Vesonder, 1991. Differential cytotoxicity and mycotoxin content among isolates of Fusarium 293-301. moniliforme. Mycopathologia 115:37-43. Wyatt, R. D., 1987. The relationship of Fusarium and
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WEIBKING ET AL. other mold produced toxins and a selected aluminosilicate in poultry. Pages D1-D15 in: Proceedings of a Symposium on Recent Developments in the Study of Mycotoxins. Kaiser Chemicals, Cleveland, OH.
Yoo, H., W. P. Norred, E. Wang, A. H. Merrill, Jr., and R. T. Riley, 1992. Fumonisin inhibition of de novo sphingolipid biosynthesis and cytotoxicity are correlated in LLC-PKi cells. Toxicol. Appl. Pharmacol. 114:9-15.
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ABSTRACT The effects of feeding Fusarium moniliforme culture material, containing known concentrations of fumonisin Bi (FBi), were studied in broiler chicks. Day-old chicks were allotted randomly to dietary treatments containing 0,1.02, 2.04, 3.06,4.08,5.10,6.12, and 7.14% fumonisin culture material (FCM). These levels of FCM supplied 0,75,150,225,300,375,450, and 525 mg of FBi /kg of feed. Each dietary treatment was fed to four pen replicates of six birds each for 21 days. Chicks fed FCM that supplied 450 and 525 mg FBi/kg diet had lower (P < .05) feed intakes and BW gains; increased (P < .05) liver and kidney weights; and increased (P < .05) mean cell hemoglobin, and mean cell hemoglobin concentrations. Compared with controls, chicks fed FCM had increased (P < .05) free sphinganine levels and sphinganine:sphingosine ratios. Treatmentassociated histological lesions were only observed in the liver of chicks fed diets containing FCM that supplied 225 mg FBi/kg or higher. Diets containing FCM that supplied levels as low as 75 mg FBi/kg affected the physiology of chicks by increasing free sphinganine levels and sphinganine:sphingosine ratios. Because inhibition of sphingolipid biosynthesis has been hypothesized as the mechanism of action of FB^ this suggests that diets containing 75 mg FBi/kg FCM may be toxic to young broiler chicks. (Key words: fumonisin Bi, broilers, Fusarium moniliforme, sphinganine, sphingosine) 1993 Poultry Science 72:456-466
reduced gains, high mortality, and development of lesions in the oral cavity Poultry rations with high levels of (Wyatt, 1987; Englehardt et al., 1989; Wu et Fusarium contamination have been as- ah, 1991). In many instances, the predomisociated with poor performance, diarrhea, nant species found was Fusarium moniliforme. There are also several reports of FMSfln'Mm-contaminated grains causing mortality in wild birds (Hagler and ParkReceived for publication July 20, 1992. hurst, 1990; Nelson et al, 1990). Accepted for publication November 24, 1992. iContribution Number 11721 of the Missouri Recently, a new group of water-soluble Agricultural Experiment Station. toxins produced by F. moniliforme has been department of Animal Sciences. characterized. These toxins, which may be 3 To whom correspondence should be addressed: S142 Animal Sciences Center, Columbia, MO 65211. responsible for the toxicological effects of 4 F. moniliforme, are collectively called Veterinary Medical Diagnostic Laboratory. INTRODUCTION
456
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Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322
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EFFECTS OF FUMONISIN ON BROILERS
MATERIALS AND METHODS
Sphlngoslne OH
OH
OH
OH
OH
Fumonisin B, (backbone)
FIGURE 1. Structures of sphinganine, sphingosine, fumonisin Bj backbone, and fumonisin Bj.
Experimental Design and Birds
One hundred and ninety-two day-old during autoclaving. Whole shelled corn female Arbor Acres x Peterson broiler (100 g) and 100 mL of distilled water were chicks were allotted randomly to pens in a added to each jar, autoclaved for 30 min at stainless steel chick battery and allowed to 121 C and a pressure of 1.4 kg/cm 2 , and consume feed and water ad libitum. The allowed to cool to room temperature. experimental design consisted of eight Fusarium moniliforme M-13255 was added to dietary treatments with four pen replicates sterilized distilled water and 2 mL of the of six birds allotted randomly to each suspension was added to the autoclaved dietary treatment. The day-old chicks were jars. A sterilized glass fiber filter was placed fed experimental diets from hatching to 21 inside the screw-on lid and the autoclave days of age. Chicks were monitored daily tape was removed from the exterior side of for signs of morbidity and mortality. the lid. The jars were shaken and lids loosened to allow for additional respiration. After 24 h of incubation at 27 C in the dark, Fumonisin Production and Analysis the jars were shaken again to insure Cultures were grown in .946-L canning complete dispersal of the fungal mycelium. jars. The lids were modified to allow more The jars were incubated in the dark for a air exchange by drilling three holes (.635- total of 5 wk at 27 C. cm) and covering them with autoclave tape A 400-mL mixture of acetonexhloroform (75:25) was added directly to each 5-wk-old culture and allowed to stand 5 Fusarium Research Center, Pennsylvania State overnight. The contents of each jar were placed in a blender for 30 s and vacuum University, University Park, PA 16802.
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fumonisins and include fumonisin Ai, A2, Bi, B2/ B3, and B4 (Gelderblom et al, 1992). Fumonisin Bj (FBi; Figure 1), the major compound, has been shown to be the causative agent in equine leukoencephalomalacia (Marasas et al, 1988) and porcine pulmonary edema (Harrison el al, 1990). Fumonisin Bi is hepatotoxic in rats (Voss et al, 1990) and has been shown to cause morphological and functional changes in chicken macrophages in vitro, which implies an immunosuppressive effect (Qureshi and Hagler, 1992). Initial studies with broilers indicated that high concentrations of FBi present in F. moniliforme cultures caused poor performance and increased organ weights, diarrhea, multifocal hepatic necrosis, biliary hyperplasia, and rickets (Brown et al, 1992; Ledoux et al, 1992). The objectives of the present study were to investigate further the toxic effects of FBi, present in F. moniliforme cultures, in young broilers and to determine the minimum dietary concentration necessary to cause deleterious effects.
458
WEIBKING ET AL.
filtered through a large buchner funnel containing fast filter paper.6 The solid culture residue was reextracted with acetonexhloroform (75:25), refiltered, and allowed to air dry overnight in the hood. The culture material was placed in a forceda ir oven at 40 C for 48 h and ground to a fine powder in a mill.7 The culture material was analyzed for FBlr FB2, and FB3 by HPLC by the procedure of Wilson et al. (1990). Diet Preparation
Sample Collection
At the end of Weeks 1, 2, and 3 of the experiment, chicks were weighed individually and feed consumption was determined for each pen. On Day 21, blood samples were collected via cardiac puncture for serum biochemistry and sphingolipid analysis and for hematologic determinations. Serum biochemical values were determined using an autoanalyzer.8 Hemoglobin was
6
Whatman International Ltd., Maidstone, England. TFitzmill Model D Comminuting machine, Fitzpatrick Co., Elmhurst, IL 60126. 8 Kodak Ektachem Analyzer, Eastman Kodak Co., Rochester, NY 14650. 9 Sigma Diagnostics, Sigma Chemical Co., St. Louis, MO 63178-9916. '"Coulter Counter Model ZB1, Coulter Electronics, Hialeah, FL 33010.
Histopathology
Post-mortem examinations were performed on six chicks from each treatment group at 3 wk of age. Chicks were anesthetized with CO2, euthanatized by cervical dislocation, and samples of proximal tibiotarsal bone, pectoral muscle, adductor muscle, ventriculus, proventriculus, lung, spleen, thymus, bursa of Fabricius, heart, duodenum, pancreas, jejunum, ileum, cecum, kidney, and liver were fixed in 10% neutral-buffered formalin. Fixed tissues were trimmed, embedded in paraffin, sectioned at 4 /«m, and stained with hematoxylin and eosin stain. All tissues from the control and from groups receiving FCM that supplied 450 and 525 mg FBi/kg were examined microscopically. Liver sections from all experimental groups were examined microscopically, liver histopathology was graded in a blind fashion, and lesion scores were reported. Statistical Analysis
Data were analyzed by the General Linear Model procedure of SAS® software (SAS Institute, 1985) as a completely randomized design. Mean differences were determined using Fisher's least significant difference test. Absolute organ weights were adjusted for final body weight by covariance analysis (Shirley, 1977). Statistical significance was accepted at P < .05. RESULTS
The effects of dietary FCM on chick performance are presented in Table 1.
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Dietary treatments were prepared by substituting ground F. moniliforme M-1325 culture material for ground corn in a typical corn-soybean meal basal diet. Fumonisin culture material (FCM) contained 7,800 mg FBj/kg by analysis and made up 0, 1.02, 2.04, 3.06, 4.08, 5.10, 6.12, and 7.14% of the respective diets and supplied 0,75,150,225, 300, 375, 450, and 525 mg FBx/kg diet, respectively. Diets were formulated to be isocaloric and isonitrogenous and either met or exceeded the nutrient requirements of broiler chicks as recommended by the National Research Council (1984). Diets and culture material were screened by the method of Rottinghaus et al. (1982) and found to be free of the following mycotoxins: anatoxin, citrinin, sterigmatocystin, zearalenone, ochratoxin A, T-2 toxin, diacetoxyscirpenol, and vomitoxin.
measured as cyanmethemoglobin.9 Red blood cell counts, mean cell volume, and hematocrits were determined with a counter10 using instrument settings described by Steel et al. (1977). Mean cell hemoglobin (MCH) and mean cell hemoglobin concentration (MCHC) were calculated. Serum sphingolipids, free sphinganine (SA), and free sphingosine (SO), were determined by HPLC using the procedures described by Merrill et al. (1988). Following blood sampling, 12 chicks per treatment were euthanatized by asphyxiation with CO2, and selected organs were excised and weighed.
459
EFFECTS OF FUMONISIN ON BROILERS TABLE 1. Effects of Fusarium moniliforme culture material, containing fumonisin, on performance of broiler chicks Culture material
Days
1 to 7
8 to 14
15 to 21
1 to 21
(r-V
(%) 0 1.02 2.04 3.06 4.08 5.10 6.12 7.14 SE
(0)2 (75) (150) (225) (300) (375) (450) (525)
109 105 108 108 107 108 99 99 3
290» 266b«i 292a 280 abcd 283 abc 287ab 261 cd
99a 100' 99a 97a 95ab 97a 88b 88b 3
232 219 231 220 226 225 212 209 6
260=*
8
461 454 460 456 459 459 445 441 6
860a 838 ab 859a 844 ab 849 a 854a 809bc 796c 13
B o d y w e i g h t g a i n (g) 1
(0) (75) (150) (225) (300) (375) (450) (525)
338 328 330 321 334 335 319 314 9
669 a 646 al * 659 a 636 al * 654 a 657 a 6181* 610c 14
Feed c o n v e r s i o n (feed:gain) (g:g) a
0 1.02 2.04 3.06 4.08 5.10 6.12 7.14 SE
(0) (75) (150) (225) (300) (375) (450) (525)
1.10' 1.05b 1.09»b 1.12' 1.12' 1.11» 1.13a l.W .02
1.25 1.22 1.27 1.27 1.25 1.27 1.23 1.24
.03
1.37 1.39 1.39 1.42 1.38 1.37 1.40 1.41 .04
1.29 1.30 1.30 1.33 1.30 1.30 1.31 1.31 .02
a_d
Values for variables within columns with no common superscripts differ significantly (P < .05). !Data are means of four replicate pens of six chicks each. 2 Dietary levels (milligrams per kilogram) of fumonisin Bj.
Compared with controls, chicks fed FCM that supplied 450 and 525 mg FBi/kg diet had reduced feed intakes at Week 2, and reduced BW gains at Week 1. Over the combined 3-wk period, chicks fed FCM that supplied 450 and 525 mg FB^/kg had reduced feed intakes and lower BW gains compared with controls. With the exception of a modest improvement in feed conversion in chicks fed FCM that supplied 75 mg FBi/kg at Week 1, feed conversion was not affected by dietary treatment. Compared with controls, increased liver weights were observed in chicks fed FCM that supplied 450 and 525 mg FBj/kg, and increased kidney weights were observed
in chicks fed FCM that supplied 375 to 525 mg FBi/kg (Table 2). Chicks fed FCM that supplied 525 mg FBi/kg had heavier proventriculus weights compared with control chicks (Table 2). Bursa of Fabricius, gizzard, and heart weights were not influenced by dietary FCM (Table 2). Compared with controls, MCH and MCHC were higher in chicks fed FCM that supplied 450 and 525 mg FBi/kg diet (Table 3). Red blood cell counts, hemoglobin, hematocrits, and mean cell volume were not influenced by dietary FCM (Table 3). With the exception of modest declines in serum cholesterol levels in chicks fed FCM that supplied 75 and 300 mg FBi/kg, serum chemistry values were
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0 1.02 2.04 3.06 4.08 5.10 6.12 7.14 SE
460
WEIBKING ET AL. TABLE 2. Effects of Fusarium moniliforme culture material, containing fumonisin, on absolute organ weights of broiler chicks1'2
Culture material
Liver
Kidney
Proventriculus
17.41** 17.21= 16.80= 16.98= 17.01= 19.03* 19.20" 19.42*
6.93=* 6.81* 7.38ocde 7.37cde 7.64t>«! 8.07* 7.86ab= 8.65* .28
4.41 b 4.55b 4.39b 4.36b 4.66b 4.85* 4.54b 5.34*
(%) 0 1.02 2.04 3.06 4.08 5.10 6.12 7.14 SE
Bursa of Fabricius
Gizzard
Heart
1.55 1.78 1.46 1.97 1.62 1.65 1.55 1.75 .13
16.01 17.39 17.79 17.49 17.47 18.10 16.95 17.78 .57
4.52 4.28 4.36 4.40 4.18 4.46 4.29 4.92 .27
(0)3 (75) (150) (225) (300) (375) (450) (525)
.59
\g)
.19
a_e
not affected by FCM (Table 4). Compared with controls, chicks fed all levels of FCM had increased free SA levels and SA:SO ratios (Figure 2). Free SO levels were not affected by dietary treatments. No lesions were seen in skeletal muscle, spleen, thymus, heart, kidney, proventriculus, lung, bursa of Fabricius, and duodenum, pancreas, jejunum, ileum, cecum, or proximal tibiotarsus of the control or treatment groups. Control and treatment groups had
incidental histologic features in the ventriculus. Mild multifocal areas of heterophil infiltration of the ventricular mucosa were associated with erosions in the koilin lining. Lesions associated with FCM treatment were observed only in the liver. A periportal granulocytic cell proliferation (Figure 3) was evident in control and treatment groups and this lesion progressed from mild to severe with
TABLE 3. Effects of Fusarium moniliforme culture material, containing fumonisin, on hematology of broiler chicks1'2 Culture material
HCT 3
(%) 0 1.02 2.04 3.06 4.08 5.10 6.12 7.14 SE
RBC
(0)3 (75) (150) (225) (300) (375) (450) (525)
a_c
(xlOVmm ) 2.59 2.66 2.50 2.60 2.49 2.47 2.44 2.48 .06
(%) 34.7 35.0 33.4 34.4 33.1 33.0 32.5 33.3 .8
HB (g/dL) 8.4 9.2 8.2 8.2 8.5 8.3 8.8 9.0 .3
MCV 3
0*m ) 127 125 127 126 126 127 126 127 1
MCH
MCHC
(Pg) 32.3b= 34.8* 32.3 bc 31.4= 34.0 abc 33.3 abc 36.0' 36.1 a 1.0
(g/dL) 24.1= 26.3 ab 24.1= 23.7= 25.5 abc 24.8b= 27.1 a 26.7 ab
Values within columns with no common superscripts differ significantly (P < .05). *Data are means of four replicates of three chicks each. 2 RBC = red blood cells; HCT = hematocrit; HB = hemoglobin; MCV = mean cell volume; MCH = mean cell hemoglobin; MCHC = mean cell hemoglobin concentration. 3 Dietary levels (milligrams per kilogram) of fumonisin B^.
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Values within columns with no common superscripts differ significantly (P < .05). 1 Absolute organ weights adjusted for final body weight by analysis of covariance. 2 Data are means of four replicates of three chicks each. 3 Dietary levels (milligrams per kilogram) of fumonisin Bj.
461
EFFECTS OF FUMONISIN ON BROILERS TABLE 4. Effects of Fusarium moniliforme culture material, containing fumonisin, on serum chemistry of broiler chicks1 Culture material
(%) 0 1.02 2.04 3.06 4.08 5.10 6.12 7.14 SE
(0)4 (75) (150) (225) (300) (375) (450) (525)
a_c
Glucose (mg/dL) 289 269 273 286 264 280 277 269 9
Total protein 3.45 3.10 3.26 3.22 3.21 3.13 3.35 3.21 .12
Albumin
- (g/dL) 1.39 1.26 1.35 1.31 1.33 1.29 1.35 1.33 .05
-
Cholesterol (mg/dL) 134^ 107< 120 abc H7abc H4bc 128ab 135* 126"'' 7
GGT2
AST3
- (IU/L) 31.5 30.3 33.9 29.1 35.0 32.5 34.2 35.9 1.8
Values within columns with no common superscripts differ significantly (P < Data are means of four replicates of two chicks each. 2 GGT = 7 glutamyl transferase. 3 AST = aspartate aminotransferase. 4 Dietary levels (milligrams per kilogram) of fumonisin Bj.
185 201 191 170 211 240 212 259 22
.05).
:
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increasing doses of FCM in the feed (Table days and resulted in poor feed conversion. 5). The cytoplasm of most of these No diarrhea was observed in the present granulocytic cells contained large spheri- study and as a result, feed conversion was cal eosinophilic granules and a large not affected by dietary treatment. In round, eccentrically located nucleus. Iso- earlier studies, FBi concentrations in FCM lated mature heterophils were also present averaged 2,000 mg/kg or less, and in in these periportal infiltrates as well as order to achieve the required dietary rare foci of erythropoiesis. These peripor- concentrations it was necessary to add tal granulocytic infiltrates were inter- significant quantities (>10%) of FCM to preted to be the extramedullary the diets. Diarrhea appears to occur only hematopoiesis. Isolated foci of hepatic when the level of FCM exceeds 10% of the necrosis with a mild heterophil and mac- diet. In the present study, the highest level rophage infiltration (Figure 3) were ob- of FCM incorporated in diets was 7.14%. served in chicks fed FCM that supplied Two possible mechanisms by which FCM 225 mg FBi/kg or greater. Moderate producing low concentrations of FBi cause diffuse hepatocellular hyperplasia was diarrhea are proposed. An additional toxic present in liver sections of chicks fed FCM metabolite may be present in F. that supplied 225 mg/kg FBi and greater. moniliforme cultures that produce low Mild biliary hyperplasia was present in concentrations of FBj. This hypothesis is isolated liver sections of chicks fed FCM supported by a recent report (Norred et that supplied 225 mg FBi/kg or greater in al, 1991) indicating the presence of other cytotoxic water-soluble metabolites in F. the ration. moniliforme cultures. A second possibility is that an unknown toxic metabolite is DISCUSSION present in all F. moniliforme cultures at a Depression in BW gains observed in low concentration, and a diarrheathis study (7 to 9%) was less than that producing dose of this metabolite is not observed in previous studies with broilers administered until greater than 10% FCM (Brown et al, 1992: 19%; Ledoux et al, is included in the ration. The increase in liver weight observed in 1992: 13%) and turkeys (Weibking et al, 1992: 19%) fed FCM that supplied 100 to this study was observed in previous 400 mg FBj/kg diet. In previous studies, a studies with broilers fed FCM that supsevere diarrhea was present after 8 to 10 plied 100 to 400 mg FBi/kg (Brown et al,
WEIBKING ET AL.
462
•
# •
Hi 150
225
300
375
450
525
600
B 150
o
a-,
CO DC
7 -
0)
c
Si
300
375
450
525
6-
o O) c x: Q. 01
,5.
0) C
.3-
c CO
225
FIGURE 3. A) Severe periportal extramedullary hematopoiesis (arrows) in liver of chick fed 525 mg fumonisin Bj/kg. Original magnification = l,000x (bar = 15 /zm). B) Hepatic necrosis (N) with mild heterophil and macrophage infiltrate in liver of chick fed 375 mg fumonisin Bj/kg. Original magnification = 200x (bar = 50 urn).
4-
.2-
O)
c .1 •
c
a.
w -75
0
75
150
225
300
Fumonisin B1
375
450
525
600
(mg/kg)
FIGURE 2. Effects of fumonisin Bj on sphinganine (A), sphingosine (B), and sphinganine: sphingosine ratio (C). Compared with controls, chicks fed diets containing fumonisin Bj had increased (P < .05) free sphinganine levels and sphinganine:sphingosine ratios.
1992; Ledoux et ah, 1992) and turkeys fed FCM that supplied 100 to 200 mg FB a /kg (Weibking et al., 1992). In contrast to broilers and turkeys, dietary FB! from FCM has been shown to decrease absolute liver weights in rats fed 93 to 139 mg FBi/ kg (Voss et al, 1990). Increases in proventriculus weights in response to FCM have also been observed previously in broilers fed FCM that supplied 100 to 400 mg FBi/ kg (Ledoux et al, 1992). Increased kidney weights observed in the present study were not seen previously in broilers fed FCM that supplied 100 to 400 mg F B ^ k g (Ledoux et al, 1992) but were seen in turkeys fed FCM that supplied 100 to 200
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75
463
EFFECTS OF FUMONISIN ON BROILERS
TABLE 5. Summary of hepatic lesions in chicks fed diets containing Fusarium moniliforme culture material with known levels of fumonisin Bj 1
Culture material
vjo esions
Periportal granulocy tic cell proliferation Severe Mild Moderate
Hepatic necrosis
Hepato-cellular hyperplasia
1/6 2/6 4/6 2/6 4/6
1/6
(%) (0)2 (75) (150) (225) (300) (375) (450) (525)
0 1.02 2.04 3.06 4.08 5.10 6.12 7.14
2/6
4/6 5/5 6/6 5/6 3/6 2/6 4/6
1/6 3/6 4/6 2/6 5/6
1/6
3/6 1/6 3/6
1
Number with trait/total. Dietary levels (milligrams per kilogram) of fumonisin Bj.
2
The failure of FCM to affect serum aspartate aminotransferase (AST) activity in the present study is surprising because increased serum AST activity has previously been observed in chicks (Ledoux et al, 1992), turkeys (Weibking et al, 1992), rats (Voss et al, 1990), and pigs (Osweiler et al, 1992) fed FBi. An increase in serum AST values is indicative of altered hepatocyte membrane integrity with the leakage of this enzyme or hepatic necrosis (Duncan and Prasse, 1986). A possible explanation for the difference between the present study and those reported previously can be found in Table 5. There was a lack of uniformity in the incidence of hepatic necrosis in chicks fed FCM that supplied 225, 300, 375, 450, and 525 mg FBi/kg. This resulted in a large standard error for mean AST values, and as a result no statistical differences were observed. It is important to note however, that individual elevated AST values are true values that correspond to individual chicks that had hepatic necrosis. Wang et al (1991), noting the remarkable structural similarity of the fumonisin backbone to SO and SA (Figure 1), hypothesized that disruption of SO metabolism might be a target of the fumonisins. In studies aimed at proving this hypothesis, Wang et al (1991) found that FBi inhibited de novo sphingolipid biosynthesis and proposed that this inhibition may be the initial molecular target of these toxins (Norred et al, 1992). Fumonisin Bj inhibition of sphingolipid biosynthesis resulted
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mg FBi/kg (Weibking et al, 1992). Other mycotoxins produced by the genus Fusarium have been reported to cause various hematological effects in broilers. Deoxynivalenol has been reported to decrease red blood cell counts and hematocrits (Huff et al, 1986), MCH (Kubena et al, 1988), and mean cell volume (Kubena et al, 1989). The T-2 toxin has been reported to cause decreased hemoglobin, mean cell volume, and MCHC (Kubena et al, 1989). In the current study, the hematological changes observed were an increase in MCH and MCHC in chicks fed FCM that supplied 450 and 525 mg FBi/kg. These mild changes are interpreted to be the result of increased red blood cell hemolysis with increasing levels of FCM in the ration. Hemolysis will cause a false elevation in MCH and MCHC because free hemoglobin will be attributed to the remaining nonhemolyzed red blood cells in the sample (Jain, 1986). Hemolysis will also result in a false decrease in red blood cell counts based on Coulter counter analysis (Jain, 1986). Both of these changes will be reflected in increased MCH and MCHC indexes. The interpretation of these results is that increased FCM in the ration may be associated with increased red blood cell fragility. It should be noted that all hematological parameters of all treatment groups were within normal ranges found in chicks Qain, 1986). Increases in MCH and MCHC were not observed in turkeys fed FCM that supplied 100 or 200 mg FBi/kg (Weibking et al, 1992).
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Results indicate that diets containing FCM that supplies 225 m g / k g or higher FBi cause hepatic lesions in young broilers. However, diets containing FCM that supplied levels as low as 75 mg FBi/kg affected the physiology of chicks by causing an increase in SA levels and SA:SO ratios. Because inhibition of sphingolipid biosynthesis has been hypothesized as the mechanism of action of FBi, this suggests that FBi toxicity may be of practical concern in broilers, as naturally occurring levels of FBi as high as 189 m g / k g have been reported in corn (Ross et al, 1991), and most broiler rations would contain a minimum of 50% corn (or 94 m g FBi/kg). The possibility that there may be other toxic metabolites in these F. moniliforme cultures that may be responsible for diarr h e a i m p l i e s t h a t F. moniliforme-
contaminated commodities may also be of concern to the broiler industry. Also of concern are recent reports indicating that in cell culture, FBi caused morphological and functional changes in chicken macrophages (Qureshi and Hagler, 1992) and was cytotoxic to turkey lymphocytes (Dombrink-Kurtzman et al, 1992). If FBi is shown to be immunosuppressive in chicks (in vivo), then levels required to compromise the immune system may well be lower than levels required to produce overt signs of toxicity. Finally, a recent report suggests that FBi may not be the only fumonisin responsible for toxicological effects. Gelderblom et al. (1992) reported that FB2 and FB3 are as toxic as FBi. If so, then the total fumonisin B levels should be used to evaluate the toxic effects of fumonisins. In the present study, total dietary fumonisin B levels (FBi + FB2 + FB3) were 0, 89,190, 283, 389, 481, 592, and 681 m g / k g , respectively. ACKNOWLEDGMENTS This study was supported in part by the National Institutes of Health Institutional Biomedical Research S u p p o r t Grant RR07053; by USDA Animal Health Formula Funding: Development of Large Scale Preparative and Analytical Methodology for the Mycotoxin, Fumonisin Bi, produced by Fusarium moniliforme; and by USDA Grant 91-37204-6684: Molecular mechanisms of animal diseases caused by fumonisin. The authors thank Paul E. Nelson, Pennsylvania State University, University Park, PA 16802 for supplying the F. moniliforme cultures; and Frank Ross and Larry Rice, National Veterinary Services Laboratories, Animal Plant Health Inspection Service, USDA, Ames, IA 50011, for confirmation and analytical assistance. REFERENCES Brown. T. P., G. E. Rottinghaus, and M. E. Williams, 1992. Fumonisin mycotoxicosis in broilers: performance and pathology. Avian Dis. 36:450-454. Dombrink-Kurtzman, G. A., G. A. Bennett, and J. L. Richard, 1992. Avian lymphocytes as an in vitro model to predict fumonisin cytotoxicity. Fed. Am. Soc. Exp. Biol. J. 6(5):A2007. Duncan, J. R., and K. W. Prasse, 1986. Veterinary
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in an accumulation of SA and an increase in the SA:SO ratio in liver hepatocytes (Wang et al, 1991), cultured renal cells (Yoo et al, 1992), and blood serum of ponies (Wang et al, 1992). Similar increases in SA levels and SA:SO ratios were observed in the present study at the lowest level of FCM that supplied 75 mg FBi/kg (Figure 2). Wang et al. (1992) hypothesized that the depletion of complex sphingolipids (resulting from FBi inhibition of biosynthesis) and accumulation of SA may well be the cause of pathology observed in FBi toxicosis, because these sphingolipids are involved in regulation of cell surface receptors, ion pumps, and other systems vital for cell function and survival. The liver lesions observed in these FBiexposed broiler chicks are similar to those reported in FBi-exposed rats, which also developed hepatocellular hyperplasia, biliary hyperplasia, and hepatic necrosis (Voss et al, 1990). The lesions observed in horses, swine, sheep, a n d b a b o o n s [encephalomalacia, pulmonary edema, acute nephrosis, and acute congestive heart failure, respectively; (Kriek et al., 1981)] were not observed in the chicks in this trial. Increased EMH was an unexpected response to increasing levels of dietary FCM. Hepatocellular hyperplasia is a likely mechanism for the increased liver weights observed in this study (Table 3).
EFFECTS OF FUMONISIN ON BROILERS
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