Growth, hematology, and histopathology of channel catfish, Ictalurus punctatus, fed toxins from Fusarium moniliforme

Aquaculture ELSEVlER

Aquaculture 130 (1995) 201-218

Growth, hematology, and histopathology of channel catfish, Ictalurus punctatus, fed toxins from Fusarium moniliforme Sonkphan Lumlertdacha”, Richard T. Lovell”,“, Richard A. Shelbyb, Stephen D. Lenz’, Barbara W. Kemppainen” “Department of Fisheries and Allied Aquacultures, Auburn University, Auburn, AL 36849, USA “Department of Plant Pathology, Auburn University, Auburn, AL 36849, USA ‘College of Veterinary Medicine, Auburn University, Auburn, AL 36849, USA

Accepted 3 1 August 1994

Abstract Year-l (average initial weight 1.2 g) and year-2 channel catfish (average initial weight 3 1 g) were fed diets containing various amounts of Fusarium monilifonne corn culture to provide 0.3 (control), 20, 80, 320, or 720 mg of fumonisin B, (FB,) /kg of diet for 10 and 14 weeks, respectively. Year-l fish fed 20 mg or more of FBI/kg of diet gained significantly less weight than the control and those fed 80 mg or more of FB, /kg of diet had significantly lower hematocrits and red and white blood cell counts than those fed lower doses. Mortality among year-l fish fed 80 mg or less of FB,/kg of diet was not significantly different from controls but over 70% of fish fed 320 or 720 mg of FB, /kg of diet died during the experiment compared to 0% in controls. Year-2 fish fed 80 mg or more of FB,/ kg of diet gained significantly less weight than fish fed lower amounts of fumonisin. Dietary concentrations of 320 mg of FBI /kg caused significantly lower hematocrit and red cell counts, and higher white cell counts. There were no significant differences in mortalities among year-2 fish fed 80 mg or less of FB,/kg of diet, but over 50% of the fish fed 320 mg or more of FB,/kg diet died from Cyfophaga columnaris infection. Fish fed the two highest doses of FB 1reduced their food consumption after 1 week and lost weight during the feeding trial. Small (2- to 4-mm diameter) white foci of subcapsular adipocyte hyperplasia were observed in the livers of year-l and year-2 channel catfish fed 20 mg or more of FB, /kg of diet. Livers of year-l and year-2 channel catfish fed 20 mg or more of FB, /kg of diet had swollen hepatocytes with lipid-containing vacuoles, lymphocyte infiltration, and scattered necrotic hepatocytes. These results indicate that diets containing Fusarium moniliforme culture material with FB, concentrations of 20 mg/kg or above are toxic to year-l and year-2 channel catfish. Kevwords: Fusarium moniliforme;

lctaluruspunctatus;

Fumonisin B,; Mycotoxins in feeds

*Corresponding author. 0044-8486/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDIOO44-8486(94)00219-3

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1. Introduction Fusarium moniliforme is recognized as one of the most prevalent fungi associated wit.h agricultural commodities, especially corn products (Marasas et al., 1984; Thiel et al., 1992 ) . Field outbreaks of disease among farm animals have been associated with consumption of corn contaminated with F. moniliforme as early as 1904 (Peters, 1904; Sheldon, 1904). .A relationship between moldy corn and equine leukoencephalomalacia (ELEM) has been known for many years (Schwarte et al., 1937; Biester et al., 1940; Binkerd et al., 1993). Recently, corn naturally infected or cultured with F. moniliforme has been fed to horses and observed to produce ELEM (Wilson et al., 1992; Ross et al., 1993). Porcine pulmonary edema (PPE) and hydrothorax were observed in swine fed corn containing F. moniliforme (Harrison et al., 1990; Colvin and Harrison, 1992; Haschek et al., 1992; Osweiler et al., 1992; Casteel et al., 1993). Chickens, ducks, and turkeys fed corn contaminated with F. moniliforme had low body weight gain, diarrhea, increased mortality and lesions in the oral cavity and liver (Engelhardt et al., 1989; Ledoux et al., 1992; Weibking et al., 1993a,b). Fumonisin B, (FB,), a water-soluble, heat-stable mycotoxin has been isolated from F. moniliforme cultures and the chemical structure identified (Gelderblom et al., 1988). Other fumonisins have subsequently been identified (Gelderblom et al., 1992) but FB, is the predominant form measured in feed samples. Fumonisin B, has been isolated from F. moniliforme cultures and administered to horses orally and by intravenous injection to produce ELEM (Marasas et al., 1988; Kellerman et al., 1990). Pulmonary edema has been produced in pigs by intravenous injection of FB, (Harrison et al., 1990; Colvin and Harrison, 1992; Haschek et al., 1992). Liver was the main target organ in rats fed a diet containing purified FB, .( Gelderblom et al., 1991). Fumonisin B, caused morphological and functional changes in chicken macrophages in vitro which indicated an immunosuppressing effect (Qureshi and Hagler, 1992). Fumonisin specifically disrupts sphingolipid metabolism (Wang et al., 1991, 1992). Elevation in the ratio of sphinganine to sphingosine has been shown to be an accurate biomarker for exposure of ponies, pigs and rats to fumonisin (Norred et al., 1992; Wang et al., 1992; Riley et al., 1993). Little information is available on the effects of F. moniliforme toxins on fish. In one study, channel catfish were fed F. moniliforme culture material containing 3 13 mg of FB , f kg of diet for 5 weeks with minimal adverse effects (Brown et al., 1994). However, absorption of FB, by channel catfish was not documented by measuring FB, or changes in sphingolipid level in channel catfish tissue. Channel catfish production is concentrated primarily in the southern part of the United States where climatic conditions of high temperature and moisture favor the growth of toxigenic fungi on agricultural crops, primarily corn (Nelson et al., 1993). Typical catfish feeds usually contain approximately 35% corn or corn screenings (Lovell, 1984). A survey of catfish feed ingredients from three feed mills in Alabama and Mississippi revealed that 80% of the corn and corn screenings sampled contained fumonisins (R.T. Lovell, unpublished data, 1993). Therefore, it was relevant to investigate the effects of feeding diets containing F. moniliforme corn culture material to channel catfish. Studies were designed to investigate the effects of feeding balanced diets containing various concentrations of fumonisins prepared with different amounts of corn

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cultured with F. moniliforme. The variables and histopathology of channel catfish.

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studied were growth, mortality,

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hematology,

2. Materials and methods Fungus culture Whole yellow corn in approximately 600-g quantities was placed into autoclavable polypropylene bags (Cole-Parmer Instrument Co., Chicago, Illinois) and soaked with 200 ml of distilled water for 2 h, then autoclaved for 1 h at 12 1°C and 120 kPa on two consecutive days. The Fusarium moniliforme strain used in these experiments was originally isolated from corn associated with ELEM in horses in Transkei, South Africa, in 1975 and deposited in the culture collection of the South African Medical Research Council as strain MRC 826. Lyophilized conidia from the F. moniliforme isolate were cultured on corn meal agar (Difco, Detroit, Michigan) plates, to which was added 1.5 ml of sterile water, at 25°C for 7 days. The fungus was transferred from the corn meal agar plates into the bags of autoclaved corn. Bags were closed by stapling and after 1 week six holes (0.6 cm diameter) were punched in the bags near the top to enhance air exchange. The bags were held at 25°C for 5 weeks, then the corn was dried in a convection oven at 50°C for 24 h, ground into meal (2 mm diameter), and stored at 4°C until used. Fumonisin analysis FB, and FB, concentrations in the cultured corn and noncultured corn were measured. A modification of the protocol of Shephard et al. ( 1990) was used for the analysis of samples containing small amounts of fumonisins, such as the noncultured corn. Ten-gram samples of ground corn were extracted with 100 ml of distilled water by shaking for 30 min. The samples were filtered and 10 ml of the filtrate was loaded onto a pre-conditioned C- 18 Seppak column (Waters Associates, Milford, Massachusetts) which was washed with 5 ml of water and the fumonisins were eluted with 2 ml of methanol, Twenty-five microliters of the eluant was derivatized with 100 ~1 of OPA reagent (o-phthaldialdehyde, 40 mg; methanol, 1 ml; 0.1 M disodium tetraborate, 5 ml; and 2-mercaptoethanol, 50 ~1). Ten microliters of the derivatized solution was injected into a high-performance liquid chromatograph (HPLC) (Waters Associates, Milford, Massachusetts) exactly 1 min after derivatization. HPLC conditions were as follows: mobile phase was 75% HPLC grade methanol and 25% 0.1 M NaH,PO, adjusted to pH 3.3 with phosphoric acid; flow rate was 2 ml/min; column was a C- 18 Novapack with a precolumn filter (Water Associates, Milford, Massachusetts) ; detection was by fluorescence at excitation and emission wavelengths of 335 and 440 nm, respectively. Quantitation of FB, and FB, was based on integration of peak areas, using Waters “Baseline” software, with subsequent linear regression of peak areas for FB, and FB, against those for standard curves determined with known concentrations of FB, and FB, standards (Sigma Chemical Company, St. Louis, Missouri). FB, and FB, have retention times of approximately 3.5 and 10.5 min, respectively. Samples with relatively high concentrations of fumonisins, such as the cultured corn, were extracted as described above and filtered first through a Whatman No. 4 filter paper and then through a 0.45 pm PTFE luer filter (Fisher Scientific Co., Pittsburgh, Pennsyl-

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vania) prior to injection into the HPLC. HPLC conditions were the same as above except that the mobile phase was 65% methanol and 0.35% 0.1 M NaH,PO, at a flow rate of 2 ml/ min. Ultraviolet absorption was monitored at 205 nm. Experimental design Diets containing various concentrations of FB, were prepared by substituting the ground corn cultured with F. moniliforme for ground, autoclaved noncultured corn in various proportions. The cultured corn contained FBI and FB, at concentrations of 1600 and 365 mg/kg, respectively. The noncultured corn contained 0.7 mg of FB, /kg. The experimental diets were formulated to contain 0.3,20, 80, 320, and 720 mg of FBI /kg of diet (Table 1). Proportions of casein, corn starch and lysine . HCl in the prepared diets were adjusted to compensate for differences in levels of nutrients between cultured corn and noncultured corn. The diets provided 32% crude protein and 3.1 kcal of digestible energy/g. Crude protein in the diet ingredients was determined by the macro-Kjeldahl method (Association of the Official Analytical Chemists, 1984) and digestible energy was estimated from tabular values for the diet ingredients for fish (National Research Council, 1983). The diets were prepared as semi-moist (30% moisture) pellets as described by El Naggar and Love11 (1991) and stored at - 18°C until fed. Table 1 Ingredient composition Ingredient

(g/kg)

Noncultured corn Cultured corn Casein Fish meal Corn starch Mineral mix’ Carboxymethylcellulose Corn oil Cod liver oil Vitamin mix* Cellulose Vitamin C3 Lysine . HCl Crude protein (%) Digestible energy (kcal/g diet) Lysine (% of protein)

of diets containing

0.3.20,

Concentration

80,320,

and 720 mg of FB,/kg

of diet

of FB, (mg/kg)

0.3

20

80

320

720

450.0 0.0 250.0 100.0 50.0 40.0 30.0 30.0 30.0 14.0 5.0 1.0 0.0 32.6

437.5 12.5 245.0 100.0 53.0 40.0 30.0 30.0 30.0 14.0 7.0 1.0 0.4 32.3

400.0 50.0 245.0 100.0 59.6 40.0 30.0 30.0 30.0 14.0 0.0 1.0 0.4 32.6

250.0 200.0 230.0 100.0 73.2 40.0 30.0 30.0 30.0 14.0 0.0 I.0 1.8 32.6

0.0 450.0 205.0 100.0 96.0 40.0 30.0 30.0 30.0 14.0 0.0 1.0 4.0 32.6

3.1 2.5

3.1 2.5

3.1 2.5

3.1 2.5

3.1 2.5

‘Williams and Briggs mineral mix (U.S. Biochemical Co., Cleveland, Ohio) was supplemented with cobalt chloride, aluminum potassium sulfide, and sodium selenite to provide 1.O, 0.7 and 0.08 mg/kg of diet, respectively. ‘The vitamin mix, diluted in cellulose, provided the following in mg/kg of diet: thiamin, 10; choline chloride, 3000; niacin, 150; riboflavin, 20; pyridoxine, 20; calcium pantothenate, 200; vitamin B-12, 0.06; retinyl acetate (500 000 IU/g), 12; alpha-tocopherol, 50; cholecalciferol (1 000 000 KU/g), 1; menadione Na-bisulfite, 80; inositol, 400; and biotin, 1. ‘Source of vitamin C was L-ascorbyl-2-phosphate-Mg (46% ascorbic acid) from Showa-Denko, Tokyo, Japan.

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Two sizes of channel catfish (ZctuZuruspunctutus), year- 1 and year-2, from the Alabama Agricultural Experiment Station were used in this experiment. The year-l fish (fry) were hatched from a single egg mass and reared in the hatchery on a starter diet for about 1 month. Subsequently, the fry, averaging 1.2 g, were stocked into 40-l aquaria (50 fish per aquarium) which were supplied with a continuous flow (2 l/min) of sand-filtered water from a lo-hectare reservoir. Water temperature was maintained at 28 k 2°C and a 14-h light/ 10-h dark photoperiod was maintained. All of the fry were fed the control diet (0.3 mg of FBI /kg of diet) for 4 days, then 50 fish were randomly assigned to each of four replicate aquaria for each of the five experimental diets. The fry were fed to satiation four times daily for 10 weeks. Each aquarium was cleaned daily. Handling procedures and facilities were in compliance with the Animal Care and Use Committee of Auburn University. The fry in each aquarium were collectively weighed weekly and examined for signs of overt toxicity. At the end of the experiment, the fish were counted, weighed collectively per replicate, and examined for signs of overt toxicity, and anesthetized with tricaine methane sulfonate (MS-222) (Argent Chemical Laboratories, Redmond, Washington) at a concentration of 50 mg/l of water for collection of blood and examination of internal organs. Blood was sampled from 11 fish from each treatment by excising the caudal peduncle and filling two heparinized microhematocrit tubes from the exposed caudal vein. The tubes were centrifuged for 10 min at 4000 X g, and hematocrits were measured. A portion of the remaining blood was used for direct counting of red blood cells and white blood cells. The blood was diluted 1: 100 with Yokoyama’s solution (Yokoyama, 1960) in a blood cell diluting pipet. The pipet was shaken for 45-60 s and transferred to a Spencer-bright-line hemacytometer, where erythrocytes and leukocytes were counted. Erythrocytes were counted in five squares and the combined number was multiplied by 5000. Leukocytes were counted in all 25 squares and the counts multiplied by 1000. Duplicate counts were made for each fish and averaged. One drop of blood from the other tube was smeared on a pre-cleaned microscope slide, smeared to a thin layer with another slide, air dried and fixed for 30 min in absolute methanol for blood cell type classification according to Grizzle and Rogers ( 1976). The blood smears were stained with Cameo Quick stain (Cambridge Chemical Products, Inc., Ft. Lauderdale, Florida), and Hemal Stain I and II (Hemal Stain Co., Inc., Danberry, Connecticut). Gill, stomach, intestine, spleen, liver, anterior kidney, posterior kidney, and brain were collected from 11 fish from each treatment and placed in Bouin’s solution for histopathological examination. The tissues were removed from the Bouin’s solution, washed in 70% ethyl alcohol until the yellow color disappeared. Preparation of tissues for visualization of lipid content was done by washing tissues in distilled water after fixation in Bouin’s solution, followed by wash with aqueous 1% lithium carbonate until the yellow color disappeared. After a 30-min rinse in distilled water, the tissues were post fixed in 1% osmium tetroxide for 24 h and rinsed in distilled water for 6 h. The tissue were embedded in paraplast for sectioning at a thickness of 5 pm. Tissues were stained with hematoxylin and eosin (H&E) (Humason, 1972) for microscopic examination. The coded slides were examined as unknowns to avoid bias in the evaluation. Year-2 channel catfish averaging 3 1 g were stocked in 70-l aquaria (30 fish per aquarium) and managed under the same conditions as described above. Thirty fish were randomly

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assigned to each of four replicate aquaria for each of the five dietary treatments described above. The fish were fed to satiation twice daily (09.00 and 17.00 h) for 14 weeks. Every 2 weeks, the fish in each aquarium were collectively weighed and examined for signs of overt toxicity. At the end of the experiment, the fish in each aquarium were collectively weighed and examined for signs of overt toxicity and anesthetized with MS-222. Blood was drawn from the caudal vein of 11 fish from each treatment by a tuberculin syringe and transferred to two heparinized microhematocrit tubes. Hematocrit, red and white blood cell counts, and differential blood cell counts were determined as described above. The abdominal cavities of the 11 fish were opened and the internal organs were examined. Liver and body weights were individually measured. Tissues were collected from 11 fish from each treatment and prepared for microscopic examination as described before. Statistical analysis Means for weight gain, survival, hematocrit, blood cell counts, and liver weight were analyzed by analysis of variance (ANOVA) to test for differences among treatments. Where ANOVA showed significant differences among treatment means, individual treatments were compared by Fisher’s least significant difference test. The analyses were performed using the SAS program (Statistical Analysis Systems, 1987). Differences were tested at the 5% level of significance.

3. Results Weight gain and survival After feeding for 2 weeks, year- 1 channel catfish fed the two highest concentrations of FB, showed a marked reduction in feed consumption and weighed significantly less than fish in other treatments (Fig. 1). By week 2, the fish fed 80 mg of FBI/kg of diet were significantly smaller than those fed lower doses, and by week 6, the fish fed 20 mg of FBI / kg of diet were smaller than the control (0.3 mg of FB, /kg of diet). After week 10, weight of the fish fed 320 mg or more of FB, /kg of diet was only approximately 6% of the control, weight of those fed 80 mg of FB, /kg of diet was 50% of the control and weight of those fed 20 mg of FB, /kg of diet was 75% of the control. Mortalities began to occur among fish fed 320 or more mg of FB,/kg of diet after feeding for 2 weeks and less than 30% of the fish in these treatments survived until the end of the IO-week experiment. Survival was 100% for the control and for fish fed 20 mg of FB,/kg of diet and 99% for fish fed 80 mg of FB, /kg of diet. Cause of mortalities was infection from Cytophuga coZumnaris bacteria as determined from examination of moribund and dead fish by the Southeastern Fish Disease Diagnostic Laboratory at Auburn University. Year-2 channel catfish fed the two highest concentrations of FBI demonstrated reduction in feeding activity after 1 week and had lost weight by week 2 (Fig. 2). By the end of week 4, weight gain was significantly lower for fish fed 80 mg of FBI/kg of diet than for the control or fish fed 20 mg of FB, /kg of diet. At the end of the 14-week feeding trial, there was no significant difference in weight gain between the control and fish fed 20 mg of FBI! kg of diet while fish fed 80 mg of FBI /kg of diet had significantly lower weight gain than these groups. Fish fed 320 and 720 mg of FBI/kg of diet had decreased feeding activity

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16 14

b

12

3 .e

‘% E .M ;

10 8 6 A

2

c 0

1

2

3

4

5

6

7

8

9

10

Weeks

Fig. I. Changes in weight gain (top) and percentage survival (bottom) during a IO-week feeding period for year1 channel catfish fed diets containing 0.3, 20, 80, 320, and 720 mg of FB,/kg of feed. Points on the curves with different letters within the same week are significantly different (P < 0.05). Means represent four replicate aquaria each initially containing 50 fish. Pooled standard error of means at weeks 1,2,3,4, 5,6,7, 8,9, and 10 are 0.05, 0.10,0.10,0.18,0.14,0.18,0.22,0.26,0.43,and0.61 forweightgainand0.32,0.32, 1.45,3.33,4.46,3.56,3.66, 3.76, 4.24, and 3.36 for survival.

and lost weight over the 14-week period. There was 100% survival during the 14-week feeding trial by fish fed 0.3, 20, or 80 mg of FB, /kg of diet, but severe mortality occurred in the groups fed 320 and 720 mg of FBI /kg of diet (Fig. 2). The mortalities occurred at week 12 through 14. The cause of mortalities was infection from C. columnaris bacteria as diagnosed by the Southeastern Fish Disease Diagnostic Laboratory. There was no difference among treatments in liver weight per 100 g of body weight. Hematology

The year- 1 channel catfish fed 320 or 720 mg of FBI /kg of diet were too small and emaciated at the end of the feeding experiment (0.4 g average weight) to collect sufficient blood sample for analysis. The lowest dietary concentration of fumonisin associated with a

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60

a

*

a

\‘i 2

4

6

8

10

12

b

14

Weeks

Fig. 2. Changes in weight gain (top) and percentage survival (bottom) during a 14-week feeding period for 2 channel catfish fed diets containing 0.3, 20, 80, 320, and 720 mg of FB,/kg. Points at a single time period different letters are significantly different (P < 0.05). Means were calculated from four replicate aquaria that each stocked with 30 fish (initially). Pooled standard error of means at weeks 2,4,6, 8, 10, 12, and 14 are 1.66,2.86,3.63,4.25,4.19, and 5.36 for weight gain and 0, 0, 0,0,0,9.06,and 12.81 for survival.

yearwith were 0.76,

significant decrease in hematocrit, red blood cell (RBC) counts and white blood cell (WBC) counts for the year- 1 fish was 80 mg of FB 1/kg of diet (Table 2). There were no morphological differences in erythrocytes or leukocytes among the various treatments as determined by microscopic observation. The lowest dietary concentration of fumonisin associated with a significant decrease in hematocrit and RBC count in year-2 fish was 320 mg of FBI/kg of diet. WBC counts were significantly higher in year-2 fish fed dietary concentrations of 320 and 720 mg of FB, /kg of diet.

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Table 2 Mean hematocrit (HT), erythrocyte (RBC), and leukocyte (WBC) counts of blood from year-l and year-2 channel catfish fed various concentrations of F. ~noniliforme culture material containing fumonisin B, (FB,)’ Dietary FB,

HT

(mg/kg)

(a)

RBC ( X IO0 cells/yl)

WBC ( X IO4 cells/pi)

36.6” 34.6” 30.7h

2.61” 2.47a 2.13”

1.29” 1.25” 0.98

38.9” 38.2” 36.3” 21.3” 26.0b

2.64” 2.61” 2.55” 1.94” 1.89”

1.32” 1.25” 1.07” 1.41b I .54b

Year-l channel catfish 0.3 20 80 Year-2 channel catfish 0.3 20 80 320 720

‘Means represent 11 fish from each treatment. Means in columns within fish year groups with the same letter are not significantly different (P > 0.05). Pooled standard error of means for HT, RBC and WBC for year- 1 channel catfish are 0.9, 0.10, and 0.05 and for year-2 channel catfish are 1.9,0.16, and 0.06, respectively.

Overt and histological signs of pathology At the end of the experiment, year- 1 channel cattish fed 80 mg of FBI /kg were markedly lighter in body color than the fish fed 0.3 or 20 mg of FB, /kg. About 20% of year-2 channel catfish in each group that were fed 80, 320 or 720 mg of FBI/kg of diet had 2- to 4-mm-

Fig. 3. Liver from year-2 channel catfish fed 80 mg of FB, /kg of diet illustrating tissue (arrow).

subcapsular

foci of adipose

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Fig :. 4. (a) Liver from year-2 channel catfish fed the control diet illustrating normal hepatocytes (H&E). Bar =:so w I. (b) Liver from year-2 channel catfish fed 20 mg of FB,/kg of diet illustrating swollen hepatocytes vfith cerItrally located nuclei and obliteration of sinusoids (H&E). Bar = SO pm.

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Fig 5. Liver from year-2 channel cattish fed 80 mg of FB,/kg (aITaws) (H&E). Bar=50 pm.

Fig. 6. Liver from year-2 channel catfish fed 20 mgof FB,/kg cytoplasmic vacuoles (arrows) (H&E). Bar = 25 pm.

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of diet illustrating

focal hepatocellular

of diet illustrating hepatocytes

net xosis

with numerous clear,

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Fig. 7. Liver from year-2 channel catfish fed 20 mg of FB,/kg of diet showing multiple foci of lipid accumul; ition (L) near the vein (arrow) (postfixed with osmium tetroxide; H&E). Bar= 500 Wm.

Fig. 8. Lymphocyte

infiltration

(arrow)

in liver of year-2 fish fed 80 mg of FB,/kg

of diet (H&E).

Bar= 50 wm

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diameter white foci of subcapsular adipocyte hyperplasia in the liver (Fig. 3)) and the liver and kidneys were pale. Livers of year-l and year-2 fish fed 0.3 or 20 mg of FBI/kg of diet appeared normal externally. Some of the year-2 fish fed 320 and 720 mg of FBI/kg of diet had livers in which the ventral portion was yellow-white. Liver was the only organ with microscopic lesions associated with consuming a diet containing F. moniliforme culture material. Lesions were found in livers of year- 1 and year2 fish fed 20 mg or more of FB 1/kg of diet. Occurrence and severity of lesions appeared to increase with concentration of fumonisins in the diet. Livers from fish fed all concentrations of fumonisins had foci of swollen hepatocytes with centrally located nuclei (Fig. 4b). Livers from year- 1 and year-2 fish fed 80 mg or more of FB, /kg of diet had foci of hepatocellular necrosis (Fig. 5) and shrunken hepatocytes. Foci of hepatocytes containing clear spherical vacuoles were observed in all fish fed fumonisins (Fig. 6). When the livers were postfixed in osmium tetroxide, the vacuoles were osmiophilic, consistent with lipid. The foci of hepatocytes containing osmiophilic vacuoles were observed near the hepatic veins in year1 and year-2 fish fed 20 mg of FB 1/kg of diet (Fig. 7) and in other areas of the liver in fish fed higher doses of fumonisins. Size and frequency of occurrence of these foci increased with dietary concentration of fumonisins. The number of osmiophilic vacuoles per hepatocyte ranged from 1 to 10, and size per vacuole ranged from 3 to 20 pm. Lymphocyte infiltrates were frequently found in areas of degenerate hepatocytes (Fig. 8).

4. Discussion Feeding balanced diets amended with corn cultured with F. moniliforme that contained as low as 20 mg of FB, /kg of diet caused growth suppression in very young (year-l) channel catfish and feeding 80 mg of FBI /kg of diet caused reduction in body weight gain in year-2 channel catfish. Liver lesions were observed in both year-l and year-2 channel catfish fed 20 mg of FBI /kg of diet. M. Li (personal communication, 1993) in a concurrent study at the Delta Research and Extension Center, Mississippi State University, Stoneville, Mississippi found that the lowest dietary concentration of fumonisin associated with depressed growth in year-2 channel catfish was 40 mg of FB, / kg of diet. The fact that reduced weight gain in year-l channel catfish was associated with a diet containing 20 mg of FBI/kg of diet, but year-2 channel catfish required higher dietary concentrations to induce reduced weight gain suggests a dose/size relationship between F. moniliforme culture material and weight gain. Channel catfish seem to be more sensitive to lower dietary concentrations of F. monilifor-me toxins than chickens and cattle, but not as sensitive as swine and horses. Cattle fed crude F. moniliforme culture material containing fumonisin at concentrations up to 148 mg of fumonisins (FBI, FB2 and FB,) /kg of diet showed no treatment-related effects on weight gain or feed consumption, although there were mild liver lesions, impaired lymphocyte function and significant differences in serum liver biochemistry (Osweiler et al., 1993). Day-old broiler chickens fed F. moniliforme culture material for 21 days showed depressed growth only when fed 450 mg of FB, /kg of diet, but showed an increase in sphinganinel sphingosine ratio when fed a diet containing only 75 mg of FBI /kg of diet ( Weibking et al., 1993a). Swine fed diets naturally contaminated with F. moniliformk and containing 155

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mg of FB, f kg died with signs of pulmonary edema and hydrothorax after 7 days of feeding (Harrison et al., 1990). Bane et al. ( 1992) provided data that showed a strong association between mystery swine disease (prenatal and neonatal mortalities) and feeding fumonisincontaminated feed containing 20 mg or more of fumonisin (Bi and B2) /kg of diet. Ross et al. ( 1992) reported that fumonisin concentrations greater than 10 mg of FBI/kg in horse feeds could cause ELEM. It is not known whether the dramatic decrease in feed consumption by fish of both sizes fed diets containing 320 and 720 mg of FBI /kg of diet was caused by toxicity or reduced palatability from the corn culture material. The fish accepted these diets initially, which suggests that they were palatable. Year-l and year-2 fish fed 80 mg of FBI/kg of diet consumed the diets satisfactorily even though showing depressed growth and liver lesions. Chickens fed corn contaminated with F. monilifonne reduced their feed consumption after feeding for 1 week when the dietary concentration of FB, was 450 mg of diet but not when metabolite may be fed lower doses (Weibking et al., 1993a). An appetite-suppressing present in the F. moniliforme corn culture material. Smith and MacDonald ( 199 1) suggested that fusaric acid affected the appetite center of swine. Boyd et al. ( 1988) reported a similar conclusion when purified T-2 (another Fusarium toxin) was fed to rats. A mechanism of action of fumonisins in animals has been hypothesized to be inhibition of sphingolipid biosynthesis. Increased ratios of sphinganine to sphingosine have been demonstrated in chickens (Weibking et al., 1993a), ponies (Wang et al., 1992)) pigs (Riley et al., 1993), and rats (Norred et al., 1992). Goel et al. (1994) measured sphingolipids in channel catfish dosed with F. moniliforme culture material as described in this study. They found that the lowest dietary concentrations of fumonisin associated with significantly elevated ratios of sphinganine to sphingosine in kidney, serum, liver and muscle were 10, 20,40, and 80 mg of FB,/kg of diet, respectively. F. moniliforme culture material does not seem to have serious effects on hematology of channel catfish. Year- 1 fish fed 80 mg of FB, /kg of diet showed a slight but significant reduction in hematocrit and red blood cell counts; however, these blood measurements were within the normal range of healthy channel catfish (Grizzle and Rogers, 1976). The blood measurements of year-2 fish were not affected even at the highest doses. Osweiler et al. ( 1993) found no significant change in either total blood cell count or differential leukocyte count of calves fed fumonisin-contaminated feed at levels up to 148 mg of FBI/kg of diet. Weibking et al. (1993a) found no change in red blood cell concentration in chickens fed fumonisin culture material providing up to 525 mg of FB, /kg of diet. Leukocyte concentration of the blood was not affected at fumonisin concentrations of 80 mg of FBI/kg of diet and below. A concurrent study (Lumlertdacha, 1994) showed that year-2 channel catfish fed 80 mg of FB, /kg of diet had a significantly higher mortality rate than control fish subsequent to challenge with Edwardsiella ictaluri (which causes enteric septicemia in channel catfish). Osweiler et al. ( 1993) reported that calves fed fumonisincontaminated feed containing 148 mg of FB,/kg of diet had impaired lymphocyte blastogenesis and elevated neutrophil antibody-dependent cytotoxicity. Liver is apparently the primary target organ for toxicity caused by F. moniliforme culture material in channel catfish. Brown et al. (1994) found no lesions in liver of adult channel catfish fed culture material containing 3 13 mg of FBI /kg of diet for 5 weeks. Hyperplastic nodules were prominent in the livers of rats (Wilson et al., 1985) and pigs (Casteel et al.,

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1993) fed fumonisin-contaminated corn. Hyperplastic nodules were not observed in the channel catfish; however, foci of subcapsular adipocyte hyperplasia were observed on the liver surface. Perhaps longer feeding would have produced hepatic nodules; Halver ( 1969) found nodules on livers of rainbow trout fed subacute doses of aflatoxin after 20 months. Areas of vacuolated hepatocytes, similar to those observed in this study, have been reported for rats (Wilson et al., 1985)) pigs (Casteel et al., 1993; Colvin et al., 1993), and horses (Ross et al., 1993) fed fumonisins, but the vacuoles were not characterized. Osmium staining indicated that these vacuoles in channel catfish hepatocytes contain lipid, which is suggestive of impaired lipid metabolism. F. moniliforme toxins induced histological changes in the catfish livers [including abnormally shaped hepatocytes (ranging from swollen to shrunken), eosinophilic cytoplasm, lymphocyte infiltration, and necrosis] in this study that were similar to histopathologic changes reported in livers of chickens (Weibking et al., 1993a), ponies (Ross et al., 1993)) and swine (Colvin et al., 1993) fed fumonisin culture material. There were no lesions in the central nervous or respiratory systems of channel catfish as were found in horses with ELEM (Marasas et al., 1988) or pigs with PPE (Colvin and Harrison, 1992; Hascheck et al., 1992; Osweiler et al., 1992) that had been fed F. moniliforme culture material. The high incidence of liver lesions in fish fed 320 and 720 mg/kg of F. moniliforme toxin coincided with a high rate of mortality from Cytophaga infection; however, these lesions were not consistent with signs of columnaris infection (Plumb, 1994). Gelderblom et al. (1991) reported that rats fed purified FBI showed similar hepatic pathological changes to rats fed F. moniliforme culture material. It has not been established that FB, alone is the primary chemical in F. moniliforme culture material causing toxicity in channel catfish. Possibly other F. moniliforme metabolites such as FB2, FB3, FB4, FA,, FAp, or fusaric acid, or AAL toxins may interact with FB, to produce the toxicity. Thus, we can only conclude that feeding F. moniliforme corn culture material to channel catfish causes decreased weight gain and liver lesions, and the concentration of FB, in the diet may be a reliable indicator of this effect. Our laboratory found fumonisins in 80% of corn and corn screenings sampled from three major catfish feed manufacture plants, although the concentrations of FBI did not exceed 10 mg/kg of diet. We also established that extrusion processing of catfish feeds does not significantly reduce the concentration in feed ingredients. Because corn and corn screenings are major ingredients in channel catfish feeds and fumonisin concentrations of 20 to 80 mg of FB,/kg are toxic to channel catfish, these ingredients should be tested for fumonisins before being used in commercial feeds.

Acknowledgements This research was supported in part by USDA grant # 92-34123-7527. We are grateful to Dr. Charles Bacon (USDA, Athens, GA) for supplying F. monilifomze isolate MRC 826.

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