IMMUNOLOGY Effects of Fumonisin B1 on ... - Semantic Scholar

IMMUNOLOGY Effects of Fumonisin B1 on Selected Immune Responses in Broiler Chicks Y. C. LI,* D. R. LEDOUX,*,1 A. J. BERMUDEZ,† K. L. FRITSCHE,* and G. E. ROTTINGHAUS† *Department of Animal Sciences, College of Agriculture, and †Veterinary Medical Diagnostic Laboratory, College of Veterinary Medicine, University of Missouri, Columbia, Missouri 65211 ABSTRACT Three experiments were conducted to evaluate immune responses in chicks fed fumonisin B1 (FB1). Day-old male chicks were randomly allotted to dietary treatments: 0, 50, 100, or 200 mg FB1/kg diet. In Experiment 1, chicks were fed diets for 3 wk and were injected intravenously with 4.6 × 106 Escherichia coli on Day 21. Blood samples were collected at 60, 120, and 180 min postinjection, and liver, spleen, and lung were collected after 180 min. Chicks fed 200 mg FB1/kg diet had significantly higher numbers of bacterial colonies in blood, spleen, and liver (P < 0.05) than control chicks. In Experiment 2, chicks were placed on the diets for 4 wk and were injected with 0.5 mL inactivated Newcastle Disease

virus vaccine on Weeks 2 and 3 of the experiment, and primary and secondary antibody titers were measured 7 d after each injection. The secondary antibody response in chicks fed 200 mg FB1/kg diet was significantly lower (P < 0.05) than that of control chicks. In Experiment 3, lymphocyte proliferation in chicks exposed to FB1 in vivo or in vitro was determined. Results of the in vivo study showed that cell proliferation in response to mitogens was lower (P < 0.05) in chicks fed 200 mg FB1/kg diet than in control chicks. For the in vitro study, cell proliferation was lower (P < 0.05) when cells were exposed to ≥ 2.5 µg FB1/mL. Data of the current study suggested that FB1 is immunosuppressive in chicks when present in the ration at 200 mg FB1/kg diet.

(Key words: chick, fumonisin B1, bacterial clearance, antibody response, lymphocyte proliferation) 1999 Poultry Science 78:1275–1282

INTRODUCTION Fumonisin B1 (FB1) is a recently discovered mycotoxin produced by several Fusarium species. Since its identification, FB1 has been shown to be associated with the previously known diseases equine leucoencephalomalacia (Marasas et al., 1988), a fatal neurological disorder of horses, and porcine pulmonary edema (Harrison et al., 1990). This toxin has also been implicated as a carcinogen in rats (Gelderblom et al., 1991), and has been linked to esophageal cancer in humans (Sydenham et al., 1990). Fumonisin B1 has also been shown to cause feed refusal, poor growth, altered serum chemistry, and organ lesions in poultry (Ledoux et al., 1992; Weibking et al., 1993a,b; Bermudez et al., 1995). Although the toxic effects of FB1 are well established, these studies were primarily focused on performance, hematology, biochemistry, and pathology. The immune system is an important defensive mechanism against invading organisms; impaired immune functions will decrease resistance to infectious diseases. Several mycotoxins have been shown to suppress immune responses and cause immunomodulation in domes-

Received for publication November 5, 1998. Accepted for publication May 2, 1999. 1 To whom correspondence should be addressed: [email protected]

tic animals. Early studies focused primarily on the impaired immune responses associated with aflatoxicosis (Pier and Heddleston, 1970; Thaxton et al., 1974; Giambrone et al., 1978; Chang and Hamilton, 1979a,b). Continuing research has demonstrated that a variety of structurally different fungal toxins may suppress immune responses and decrease host resistance to infectious diseases (Dwivedi and Burns, 1984a,b; Pier and Mcloughlin, 1985; Corrier et al., 1987). Limited information exists with respect to the effects of FB1 on the immune system of chicks. Qureshi and Hagler (1992) reported that FB1 was cytotoxic to chick macrophages in vitro. Chick peritoneal macrophages exposed to FB1 in vitro showed morphological alterations, including nuclear disintegration and cytoplasmic blebbing. The phagocytic potential of chick peritoneal macrophages was also significantly inhibited. Cytotoxicity of FB1 was also observed in turkey lymphocytes (DombrinkKurtzman et al., 1994). Turkey lymphocytes exposed to FB1 in vitro exhibited cytoplasmic vacuolization and were not able to proliferate. In comparison to these in vitro data, there is relatively little information on the effects of FB1 on the immune system of chicks in vivo. Two studies have reported that chicks fed Fusarium proliferatum culture

Abbreviation Key: ConA = concanavalin A; cpm = counts per minute; FB1 = fumonisin B1; FCM = fumonisin culture material; LPS = lipopolysaccharide; NDV = Newcastle Disease virus; PWM = pokeweed mitogen.

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material containing FB1, FB2, and moniliformin had lowered immune responses. Dombrink-Kurtzman et al. (1993) found that lymphocyte viability in chicks fed diets containing FB1, FB2, and moniliformin was significantly decreased. Significant suppression in total Ig and IgG levels was observed in chicks fed diets containing FB1, FB2, and moniliformin (Qureshi et al., 1995). Reduction in phagocytic potential of peritoneal macrophages was also reported. Although these studies demonstrated that chicks fed diets amended with Fusarium proliferatum culture material had lower immune responses, the culture material used in both studies contained more than one class of toxin. The toxin that was responsible for the immunosuppressive effect observed in those studies remains to be determined. The objective of this study was to evaluate the immune response of chicks fed FB1. In the present study, bacterial clearance rate, antibody response to inactivated Newcastle disease vaccine, and mitogen-induced lymphocyte proliferation were used to assess immune functions of chicks fed FB1.

MATERIALS AND METHODS Birds and Diets One-day-old Arbor Acres × Arbor Acres male broiler chicks were used in all experiments.2 Chicks were individually weighed, wing-banded, and randomly assigned to pens in a stainless steel battery. Chicks were maintained on a 24-h constant-light schedule and allowed to consume feed and water ad libitum. Four dietary treatments containing either 0, 50, 100, or 200 mg FB1/kg of diet were used in this study. Dietary treatments containing FB1 were prepared by substituting ground Fusarium moniliforme culture material (FCM), which contained 6,520 mg FB1/ kg culture material, for ground corn in a typical cornsoybean meal basal diet. Diets were formulated to be isocaloric, isonitrogenous, and either met or exceeded the nutrient requirements of broiler chicks as recommended by the National Research Council (1994). Diets were screened for the presence of mycotoxins by the method of Rottinghaus et al. (1982) and were found to be free of aflatoxin, citrinin, vomitoxin, sterigmatocystin, zearalenone, ochratoxin A, and moniliformin. Chicks were individually weighed, and feed intake and feed efficiency was determined for each pen on a weekly basis in order to monitor growth of chicks fed different diets. Chicks were checked daily for signs of disease and mortality. The animal care and use protocol was reviewed and approved by the University of Missouri-Columbia Animal Care and Use Committee.

Bacterial Clearance Rate An Escherichia coli challenge was employed to evaluate the ability of chicks fed FB1 to clear bacteria from circula2

Stover Hatchery, Stover, MO 65078. Remel, Lenexa, KS 66215. 4 SOLVAY Animal Health, Inc., Mendota Heights, MN 55120. 5 IDEXX Laboratory, Inc., Westbrook, ME 04029.

tion. This E. coli originated from a case of colibacillosis in commercial turkeys. One-day-old male broiler chicks were randomly assigned to one of four dietary treatments with six replicates of two chicks per treatment for 3 wk. On Day 21, chicks were intravenously injected with 4 × 106 E. coli. The amount of E. coli used was based on pilot experiments, in which the clearance of different doses of E. coli (106, 107, 108) was investigated. For this study, a dose was chosen that showed complete elimination of bacteria from circulation but did not cause severe hemodynamic changes influencing clearance function. A dose of 1 × 106 E. coli was completely eliminated from the circulation during a time period between 60 and 120 min in chicks. At a higher dose (108), delayed clearance of E. coli was observed with chicks unable to completely clear the bacteria by 180 min after inoculation. Four milliliters of blood was collected from the jugular vein into heparinized tubes at 60, 120, and 180 min postinjection. Prior to E. coli inoculation, blood samples from each bird were collected and analyzed for the presence of E. coli in order to insure no preexisting infections. At 180 min after bacterial injection, chicks were euthanatized and tissue samples of liver, spleen, and lung were taken for quantitative bacterial determination. Whole blood samples were serially diluted with sterile PBS, and 100 µL of each dilution at each time point was plated onto MacConkey agar plates.3 Organs were weighed, and approximately 1 g of tissue samples was homogenized with 5 mL of sterile PBS. Serial dilutions of tissue suspension (100 µL) were plated onto MacConkey agar plates. Each sample was plated in duplicate. Plates were incubated at 37 C for 18 h, and the E. coli colony-forming units were enumerated. The colonies grown on the plates were confirmed to be E. coli colonies by previously reported methods (Patten et al., 1995). The final bacterial concentrations were calculated as the numbers of colony-forming units per milliliter of blood and as colony-forming units per gram of harvested tissue.

Antibody Response Primary and secondary antibody response to inactivated Newcastle Disease virus (NDV) vaccine was used to evaluate the humoral immunity of chicks. Forty-eight 1-d-old male chicks were randomly assigned to one of four dietary treatments (six replicates of two chicks each per treatment) for 4 wk. At the end of the 2nd and 3rd wk of the experiment, chicks were injected intramuscularly with 0.5 mL of a commercial inactivated NDV vaccine.4 Blood samples were withdrawn from the jugular vein 7 d after each injection for determination of the primary and secondary antibody response. Serum samples were harvested and stored at −20 C until analysis. Antibody titers to inactivated NDV were measured by an ELISA. The ELISA was performed with commercial kits according to manufacturer’s recommendations.5

Lymphocyte Proliferation

3

In Vivo Study. Lymphocyte proliferative response in chicks fed FB1 was evaluated in this study. One-day-old

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male chicks were randomly assigned to one of two dietary treatments, control or 200 mg FB1/kg of diet. Each of the two diets were fed to four pens of four chicks per pen for 3 wk. A [3H]-thymidine uptake assay was used to assess the proliferation of chick lymphocytes in response to three polyclonal mitogens, concanavalin A (ConA), pokeweed mitogen (PWM), and E. coli O26:B6 lipopolysaccharide (LPS). Three mitogens were chosen so that proliferative response of T and B cells can be distinguished. Concanavalin A is known to stimulate T cell proliferation, and bacterial LPS selectively stimulates B cell proliferation. However, it has been reported that chick lymphocytes respond poorly to LPS (Hovi et al., 1978; Fritsche et al., 1991). Thus, PWM, which induces a strong B cell response, was also chosen. All mitogens were purchased from Sigma Chemical Co.6 At the end of the feeding trial, lymphocytes were isolated from peripheral blood using Lymphocyte Separation Medium (density 1.077) as described by the manufacturer.6 Cells were adjusted to a final concentration of 2 × 107 cells/mL in RPMI-1640 medium. One hundred microliters of cell suspension (2 × 106 cells) was added to each well in 96-well microtiter plates. Cells were incubated with optimal concentrations of either ConA (20 µg/mL), PWM (10 µg/mL), or LPS (5 µg/mL) in the presence of homologous chick serum (5%). Cell cultures were incubated at 40 C for 48 h in a humidified incubator with 5% CO2. For the final 16 h incubation, 1 µCi of [3H]thymidine in 10 µL of RPMI-1640 medium was added to each well.7 Upon completion of incubation, cells were harvested8 and radioactivity in each well was counted in a liquid scintillation counter.9 Each sample was tested in triplicate wells and the values were averaged and expressed as mean counts per minute (cpm). A stimulation index, which was calculated as the mean CPM of tritium incorporated in cells treated with mitogens divided by the mean CPM of tritium incorporated in cells not treated with mitogens, was also calculated. In Vitro Study. Three-week-old male chicks were used to determine the mitogenic response of chick lymphocytes exposed to FB1 in vitro. Purified FB1 was purchased from Sigma Chemical Co.10 Various concentrations of FB1 (0 to 10 µg/mL) were prepared and were incubated with lymphocytes (2 × 106) in the presence of ConA (20 µg/ mL) for 40 h. Procedures used in this study to quantify lymphoproliferation were the same as described in the in vivo study.

Statistical Analysis Data were subjected to one-way ANOVA and the means for treatments showing significant differences in

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Histopaque威-1077, Sigma Chemical Co., St. Louis, MO 63178-9916. Amersham Corp., Arlington Heights, IL 60005. PHD Harvester, Cambridge Technology, Inc., Watertown, MA 02172. 9 Beckman Instruments, Inc., Schaumburg, IL 60173-3833. 10 Sigma Chemical Co., St. Louis, MO 63178-9916. 11 Abacus Concepts, Inc., Berkeley, CA 94701. 7 8

TABLE 1. Effects of dietary fumonisin B1 (FB1) on feed intake, body weight gain, and feed conversion in broiler chicks1,2 Experiment 1

2

3

FB1 level

Feed intake

Body weight gain

Feed:gain

0 50 100 200 Pooled SE 0 50 100 200 Pooled SE 0 200 Pooled SE

876 921 954 878 34 1,663 1,733 1,663 1,709 51 816 784 17

(g) 718 733 759 693 25 1,145 1,196 1,167 1,131 35 620 599 10

(g:g) 1.22 1.26 1.26 1.26 0.02 1.45 1.45 1.42 1.51 0.07 1.30 1.29 0.02

1 Data are means of six pen replicates of two chicks each (Experiments 1 and 2) or four pens of four chicks each (Experiment 3). 2 The length of study was 21 d for Experiments 1 and 3 and 28 d for Experiment 2.

the ANOVA were compared by Fisher’s protected least significant difference procedure (Snedecor and Cochran, 1967). Significance was accepted at P < 0.05. The data for bacterial clearance and antibody titer were logarithmic transformed prior to analyses in order to achieve homogeneity of variance. All analyses were conducted on a Macintosh computer using SuperANOVA.11

RESULTS AND DISCUSSION Growth Response The effects of dietary FB1 on feed intake, body weight gain, and feed conversion are summarized in Table 1. Over the total 3-wk period in the Experiments 1 and 3 and the 4 wk period in the Experiment 2, chicks fed diets containing FB1 performed as well as those fed the control diet. Feed intake and body weight gain of chicks fed FB1 did not differ significantly (P > 0.05) from those of control chicks. Feed conversion was also not affected (P > 0.05) by dietary FB1. In the present study, results of performance were consistent with those of Weibking et al. (1993a), who reported that body weight gain, feed intake, and feed conversion in chicks fed 375 mg FB1/kg or less supplied by FCM did not differ significantly from values of chicks fed the control diet. In contrast, an earlier study conducted by Ledoux et al. (1992) showed a linear depression in weight gain in chicks fed FCM that supplied 100 to 400 mg FB1/kg diet. Differences in the present study and the study of Weibking et al. (1993a) with that of Ledoux et al. (1992) may be related to the concentration of FCM incorporated into the diets. In the earlier study, because of low concentrations of FB1 in FCM, large amounts of FCM needed to be added to the diets in order to achieve the required FB1 levels. A depression in dry matter digestibility related to diarrhea was observed in the earlier study (Ledoux et al., 1992). It was suggested that depressed weight gain with diarrhea was a result of

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LI ET AL. TABLE 2. Effect of fumonisin B1 (FB1) on Escherichia coli clearance from blood in chicks1 Time postinjection FB1

60 min

(mg/kg diet) 0 50 100 200

2.87 2.91 2.99 3.17

± ± ± ±

120 min 0.03a,z 0.05a,yz 0.04a,y 0.06a,x

180 min

(log10 cfu/mL blood)2,3 2.35 ± 0.06b,z 2.41 ± 0.10b,z 2.89 ± 0.04a,y 3.16 ± 0.01a,x

Source of variation Treatment Time Treatment × time

1.08 1.13 1.50 2.88

± ± ± ±

Pooled mean

0.23c,y 0.25c,y 0.10b,y 0.11b,x

2.10 2.15 2.46 3.07

± ± ± ±

0.19y 0.20y 0.17y 0.05x

P 0.001 0.001 0.002

Values within a row with no common superscript letter differ (P < 0.05). Values within columns with no common superscript differ (P < 0.05). 1 Data are represented as mean ± SE of six pen replicates of two chicks each. 2 Whole blood samples were serially diluted with sterile PBS, and 100 µL diluted samples were placed onto MacConkey agar plates and incubated at 37 C for 18 h. The number of E. coli cfu were determined. 3 Escherichia coli colonies were logarithmic-transformed to achieve homogeneity of variance and were expressed as log10 cfu. a–c

x–z

incorporation of high levels of FCM ( 10%) in the diets. Neither poor performance nor diarrhea was observed in the present study and in that of Weibking et al. (1993a). In both studies, levels of FCM (< 5%) incorporated into the diets were much lower. In addition, in the present study, no signs of illness and mortality were observed throughout the feeding trial.

Bacterial Clearance Rate The effects of dietary FB1 on bacterial clearance from circulation is shown in Table 2. Microbiological cultures from blood samples taken before E. coli injection were sterile in all groups. Bacterial clearance from the circulating blood was significantly affected by dietary FB1. Significantly delayed systemic clearance (P < 0.05) with persisting numbers of E. coli in the blood until the end of the observation period was seen in chicks fed 200 mg FB1/kg diet as compared to chicks fed either control, 50, or 100 mg FB1/kg diet. Compared with control chicks, chicks fed 100 mg FB1/kg diet had significantly higher (P < 0.05) numbers of E. coli colony-forming units in the blood samples at 60 and 120 min postinoculation. In contrast to this, blood cultures of some chicks in control (6/ 12) and 50 mg FB1/kg diet (5/12) groups were sterile at 180 min after inoculation. Systemic bacterial clearance was also time-dependent (Table 2). The numbers of E. coli colony-forming units were reduced with increasing postinjection time in all groups. However, a significant interaction of treatment by time was observed, indicating that the ability of chicks to eliminate bacteria from the blood system was diminished by dietary FB1. Data showed that E. coli in chicks fed the control diet were cleared at a faster rate (P < 0.05) than those in chicks fed 200 mg FB1/kg diet (Table 2). Compared with control chicks, the delayed systemic bacterial clearance in the chicks fed 200 mg FB1/kg diet was accompanied by significantly higher (P < 0.05) numbers of E. coli in the liver

and spleen (Table 3). Significantly higher (P < 0.05) numbers of E. coli colony-forming units were also observed in spleen samples in chicks fed 100 mg FB1/kg diet. Data of the current study indicate that diminished systemic bacterial clearance was associated with a significant increase of E. coli in tissues, suggesting both reduced phagocytosis and lysis capacity of the reticuloendothelial system. The first step of the host defense against bacterial infection is phagocytosis and degradation of bacteria by phagocytic cells. If the phagocytic system is compromised, the ability to resist infection is likely decreased. Delayed bacterial clearance from the blood system associated with reduction in phagocytic potential of heterophils and macrophages has been shown in chicks exposed to aflatoxin (Chang and Hamilton, 1979a,b; Neldon-Ortiz and Qureshi, 1992). Dietary FB1 may also impair phagocytosis by heterophils or macrophages, thus leading to a reduction in bacterial clearance during infections. In a previous study, Qureshi and Hagler (1992) reported that

TABLE 3. Counts of viable bacteria in organs of chicks fed diets contaminated with Fumonisin B1 (FB1)1 FB1

Spleen

(mg/kg diet) 0 50 100 200 Pooled SE

4.94c 5.16bc 5.33b 5.77a 0.12

Liver (log10 cfu/g tissue)2,3 4.61b 4.51b 4.58b 4.78a 0.06

Lung 3.01a 3.04a 3.02a 2.99a 0.13

a–c Values within columns with no common superscript differ significantly (P < 0.05). 1 Data are means of six pen replicates of two chicks each. 2 Approximately 1 g of tissue samples were homogenized with 5 mL of sterile PBS. Tissue homogenate was serially diluted with sterile PBS and 100 µL of diluted tissue suspension were placed onto MacConkey agar plates and incubated at 37 C for 18 h. The number of E. coli colonyforming units were determined. 3 Escherichia coli colonies were logarithmic transformed in order to achieve homogeneity of variance, and were expressed as log10 cfu.


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TABLE 4. Primary and secondary antibody response to inactivated Newcastle Disease vaccine in chicks fed Fumonisin B1 (FB1)1 FB1

Primary antibody titer (log10)2

(mg/kg diet) 0 50 100 200 Pooled SE

Secondary antibody titer

a

3.57 3.47a 3.33a 3.50a 0.11

4.40a 4.35a 4.32a 4.13b 0.05

a,b Values within columns with no common superscript differ (P < 0.05). 1 Data are means of six pen replicates of two chicks each. 2 Antibody titers were logarithmic-transformed to achieve homogeneity of variance and were expressed as log10 antibody titer.

FB1 was cytotoxic to chick macrophages and caused depression in the phagocytic potential of chick peritoneal macrophages. In addition, chicks fed diet amended with F. proliferatum culture material, containing 61 mg FB1/ kg, 10.5 mg FB2/kg, and 42.7 mg moniliformin/kg, had reduced numbers of peritoneal macrophages (Qureshi et al., 1995). Macrophages from toxin-fed birds also exhibited a reduction in phagocytic potential. The findings of these studies imply that chicks exposed to Fusarium toxins may be more susceptible to infectious diseases, resulting from dysfunction of the mononuclear phagocytic system. Furthermore, Gelderblom et al. (1988) reported that dietary FB1 caused necrosis in liver cells including Kupffer cells (hepatic macrophages), suggesting an alteration in cell function. Apart from impaired phagocytosis of macrophages, reduction in bacterial clearance as a consequence of decreased phagocytic and lytic potential of heterophils can not be ruled out.

Antibody Response to Inactivated Newcastle Disease Virus Table 4 shows the primary and secondary antibody response to inactivated NDV in chicks fed FB1. Dietary FB1 did not significantly affect the primary antibody response to inactivated NDV. A similar observation was reported by Weibking et al. (1994), who found that primary antibody response to SRBC was not affected in chicks fed FB1 for 3 wk. The current study showed that secondary antibody titers against killed NDV in chicks fed 200 mg FB1/kg diet were significantly lower (P < 0.05) than those in chicks fed control, 50, or 100 mg FB1/kg diet. Suppressed humoral immunity was also observed in previous studies. Martinova (1996) reported that mice injected with a single dose of FB1 (0.25 to 2.5 mg/kg body weight) and simultaneously with a suboptimal dose of SRBC had reduced numbers of plaque-forming cells. Significant suppression in total Ig and IgG levels in primary and secondary response to SRBC was observed in White Leghorn chicks fed diets containing 61 mg FB1/kg, 10.5 mg FB2/kg, and 42.7 mg moniliformin/kg for 6 wk (Qureshi et al., 1995). In contrast to the findings of Qureshi et al. (1995), primary antibody response to inactivated NDV

FIGURE 1. Effect of dietary fumonisin B1 on chick lymphocyte proliferation in response to mitogens [20 µg concanavalin A (ConA)/mL, 10 µg pokeweed mitogen (PWM)/mL, and 5 µg lipopolysaccharide (LPS)/ mL]. Data are expressed as mean stimulation index with error bars representing the standard error. Each value is the mean of four pen replicates of four chicks each. Means that do not share a common letter are different (P < 0.05). *Stimulation index is calculated as the mean counts per minute (cpm) of tritium incorporated in cells treated with mitogens divided by the mean counts per minute of tritium incorporated in cells not treated with mitogens. Radioactivity in the control wells (without mitogens) was 1,125 ± 87 cpm (m ± SE) for control chicks and 938 ± 54 cpm (m ± SE) for FB1-fed chicks.

was not affected by dietary FB1 in the current study. In addition, depressed secondary antibody response to inactivated NDV was only observed in chicks fed 200 mg FB1/kg. One explanation could be that culture material used in the previous study contained fumonisins and moniliformin, which may have interacted synergistically. It has been shown that toxicity of some individual mycotoxins can be enhanced when they are present as cocontaminants in feeds (Huff and Doerr, 1981; Huff et al., 1988). In addition, a longer feeding period (6 wk) and different type of chick (layer breed) used in previous study may have affected the outcome. Suppression of humoral immunity has been observed previously in chicks fed Fusarium culture material. Humoral immunity against SRBC was suppressed in chicks fed diets containing 2% corn infected with either F. moniliforme, Fusarium semitectum, or Fusarium equiseti (Chu et al., 1988; Wu et al., 1991). Male White Leghorn chicks have also shown decreased secondary agglutinin response to SRBC and Brucella abortus after consuming FCM for a period of 6 wk (Marijanovic et al., 1991). In those studies, the culture materials were not screened for the presence of these mycotoxins. It is known that Fusarium species are capable of producing several toxins, including fumonisins, fusarins, fusaric acid, moniliformin, trichothecenes, and other unknown toxins; the immunosuppressive effect observed in those studies could be caused by any of a number of toxins.

Mitogen-Induced Lymphocyte Proliferation Effect of dietary FB1 on chick lymphocyte proliferation is presented in Figure 1. Lymphocyte proliferation in re-

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sponse to ConA and PWM was significantly lower (P < 0.05) in chicks fed 200 mg FB1/kg diet than in control chicks. Data showed that lymphocyte proliferation in response to ConA and PWM in chicks fed FB1 was depressed by 25 and 17%, respectively. In contrast, lymphocyte proliferation in response to LPS was similar (P > 0.05) in chicks fed control and 200 mg FB1/kg. In the absence of mitogens, lymphocyte proliferation was higher in control chicks [1,125 ± 87 (SE) cpm] than in chicks fed FB1 [938 ± 54 (SE) cpm], but the difference was not significant (P > 0.05). Proliferation in response to LPS was very poor compared to that of ConA and PWM. Poor proliferative response to LPS in chick lymphocytes observed in the current study was also reported in previous studies (Hovi et al., 1978; Fritsche et al., 1991). The low levels of proliferation may relate to insensitivity of chick lymphocytes to bacterial endotoxins (Adler and Damassa, 1978). In addition, the lack of significant difference between control chicks and those fed FB1 in terms of lymphocyte proliferation in response to LPS may also relate to chick lymphocytes insensitivity to LPS. Although LPS induced only very low levels of proliferation in chick B lymphocytes, PWM, a strong B cell mitogen, stimulated a significant B cell response in chicks. The reason for using three mitogens in the current study was to be able to distinguish the proliferative response of T and B cells. Results of the present study showed that lymphocyte proliferation in response to ConA and PWM in chicks fed FB1 were significantly lower than that of control chicks, suggesting that both T and B cell populations were affected by FB1. Dietary FB1 has also been shown to reduce lymphocyte proliferation in other species. Osweiler et al. (1993) reported a reduction in peripheral lymphocyte proliferation in response to mitogens, including ConA, PWM, and phytohemagglutinin in calves fed 148 mg FB1/kg diet for 30 d. In an earlier study, Dombrink-Kurtzman et al. (1993) observed a reduction in lymphocyte viability in chicks fed rations amended with F. proliferatum culture material containing either 61 mg FB1/kg, 14 mg FB2/kg, and 66 mg moniliformin/kg or 193 mg FB1/kg, 38 mg FB2/kg, and 193 mg moniliformin/kg. Lymphocytes from chicks exposed to toxins also exhibited vacuolated cytoplasm. Although the cytotoxicity of FB1 was not determined in the present study, one can speculate that proliferation of lymphocytes exposed to FB1 can be adversely affected if FB1 decreases cell viability. Data of FB1 exposure in vitro on lymphocyte proliferation are presented in Figure 2. Proliferation of lymphocytes declined linearly (P < 0.01) with increasing FB1 concentrations. Cells exposed to 5 µg FB1/mL showed 50% inhibition of proliferation, whereas 75% inhibition of proliferation was observed in cells exposed to 10 µg FB1/mL. A similar result was observed previously in turkey cells (Dombrink-Kurtzman et al., 1994). Turkey lymphocytes exposed to FB1 (0.01 to 25 µg/mL) for 48 and 72 h revealed a dose-dependent inhibition of cell proliferation. The levels of FB1 that caused 50% inhibition of proliferation after 48 and 72 h exposure were 5 and 1.4

FIGURE 2. Mitogenic response of chick peripheral lymphocyte exposed to fumonisin B1 in vitro and co-cultured with 20 µg concanavalin (ConA)/mL. Data are expressed as mean stimulation index with error bars representing the standard error. Each value is the mean of 12 chicks per dose. Means that do not share a common letter are different (P < 0.05). Linear and quadratic effects of fumonisin B1 were also observed (P < 0.01). *Stimulation index is calculated as the mean counts per minute (cpm) of tritium incorporated in cells treated with ConA divided by the mean counts per minute of tritium incorporated in cells not treated with ConA. Radioactivity in the control wells (without ConA) was 1,126 ± 38 cpm (m ± SE).

µg/mL, respectively. Fumonisin B1 has also been shown to reduce responses to mitogenic stimulation in mouse spleen and thymus cells (Martinova, 1996). [3H]-thymidine incorporation into mouse spleen T and B cells exposed to mitogens was significantly decreased in the presence of FB1, but the stimulation was not completely blocked. The mechanism of action of FB1 has been shown to involve the disruption of sphingolipid biosynthesis, leading to depletion of complex sphingolipids and accumulation of free sphinganine and sphingosine (Wang et al., 1991). The immunological effects of FB1 observed in the present study may relate to the depletion of complex sphingolipids and accumulation of sphingolipid breakdown products. Inhibition of sphingolipid synthesis by FB1 was correlated with suppressed cell proliferation (Wang et al., 1991; Yoo et al., 1992). Sphingolipid breakdown products have also been recognized as anti-proliferative and tumor-suppressor lipids (Felding-Habermann et al., 1990; Hannun and Linardic, 1993), and were demonstrated to inhibit humoral and cell-mediated immune responses (Martinova, 1996). Although the levels of sphinganine and sphingosine in chicks fed FB1 were not determined in the current study, previous work in our laboratory showed that chicks fed a diet containing FB1 as low as 75 mg/kg had increased free sphinganine and sphingosine levels. Thus, consumption of FB1 could result in impaired immune function, presumably via disruption of sphingolipid metabolism. In conclusion, the objective of the current research was to determine whether dietary FB1 adversely affects the immune response in chicks. The results showed that dietary FB1 decreased humoral immunity, suppressed lymphocyte proliferation, and reduced bacterial clearance.


Furthermore, neither depressed performance nor signs of illness were observed in chicks fed FB1 in the current study. These findings suggest that chicks may be immunosuppressed without any overt sign of toxicosis during exposure to the fusarium toxin. The levels of dietary FB1 causing immunosuppression are relatively high (200 mg/ kg) as compared to the naturally occurring levels of FB1, 0.1 to 10 mg/kg, in world’s corn supply. The lowest level of FB1 used in this study is five times higher than the naturally occurring levels of FB1, and at this level, FB1 did not have a negative effect on the immune system. Therefore, FB1 by itself may not be a threat to the poultry industry in terms of impaired immune function. However, in commercial poultry production chicks are constantly exposed to a variety of stressors that could adversely affect the immune system. Consumption of FB1 together with these stressors may put the immune system at an added risk. In addition, a typical poultry ration is made up of several grain sources; each of which may be contaminated with a different mycotoxin or more than one mycotoxin. Thus, other mycotoxins in addition to FB1 may be present simultaneously in a poultry ration. Toxicity of FB1 has been shown to be enhanced when it is present with other mycotoxins as co-contaminants in feeds (Javed et al., 1993; Kubena et al., 1995), and in this context, FB1 could be a potential concern to the broiler industry.

REFERENCES Adler, H. E., and A. J. Damassa, 1978. Toxicity of endotoxins to chicks. Avian Dis. 23:174–178. Bermudez, A. J., D. R. Ledoux, and G. E. Rottinghaus, 1995. Effects of Fusarium moniliforme culture material containing known levels of fumonisin B1 in ducklings. Avian Dis. 39:879–886. Chang, C. F., and P. B. Hamilton, 1979a. Impaired phagocytosis by heterophil from chickens during aflatoxicosis. Poultry Sci. 48:459–466. Chang, C. F., and P. B. Hamilton, 1979b. Impairment of phagocytosis in chicken monocytes during aflatoxicosis. Poultry Sci. 58:562–566. Chu, Q., M. E. Cook, W. Wu, and E. B. Smalley, 1988. Immune and bone properties of chicks consuming corn contaminated with a Fusarium that induces dyschondroplasia. Avian Dis. 32:132–136. Corrier, D. E., P. S. Holt, and H. H. Mollenhauer, 1987. Regulation of murine macrophage phagocytosis of sheep erythrocytes by T-2 toxin. Am. J. Vet. Res. 48:1304–1307. Dombrink-Kurtzman, M. A., G. A. Bennett, and J. L. Richard, 1994. An optimized MTT bioassay for determination of cytotoxicity of fumonisins in turkey lymphocytes. J. AOAC Int. 77:512–516. Dombrink-Kurtzman, M. A., T. Javed, G. A. Bennett, J. L. Richard, L. M. Cote, and W. B. Buck, 1993. Lymphocyte cytotoxicity and erythrocytic abnormalities induced in broiler chicks by fumonisins B1, B2, and moniliformin from Fusarium proliferatum. Mycopathologia 124:47–54. Dwivedi, P., and R. B. Burns, 1984a. Pathology of ochratoxin A in young broiler chicks. Res. Vet. Sci. 36:92–103. Dwivedi, P., and R. B. Burns, 1984b. Effect of ochratoxin A on immunoglobulins in broiler chicks. Res. Vet. Sci. 36:117–121. Felding-Habermann, B., Y. Igarashi, B. A. Fenderson, L. S. Park, N. S. Radin, J. Inokuchi, G. Strassmann, K. Handa, and S. Hakomori, 1990. A ceramide analogue inhibits T cell prolifer-

1281

ative response through inhibition of glycosphingolipid synthesis and enhancement of N,N-dimethylsphingosine synthesis. Biochem. J. 29:6314–6322. Fritsche, K. L., N. A. Cassity, and S. Huang, 1991. Effect of dietary fat source on antibody production and lymphocyte proliferation in chickens. Poultry Sci. 70:611–617. Gelderblom, W.C.A., K. Jaskiewicz, W.F.O. Marasas, P. G. Thiel, R. M. Horak, R. Vleggaar, and N.P.J. Kriek, 1988. Fumonisinsnovel mycotoxins with cancer-promoting activity produced by Fusarium moniliforme. Appl. Environ. Microbiol. 54:1806–1811. Gelderblom, W.C.A., N.P.J. Kriek, W.F.O. Marasas, and P. G. Thiel, 1991. Toxicity and carcinogenicity of the Fusarium moniliforme metabolite, fumonisin B1, in rats. Carcinogenesis 7:1247–1251. Giambrone, J. J., D. L. Ewert, R. D. Wyatt, and C. S. Eidson, 1978. Effect of aflatoxin on the humoral and cell-mediated immune systems of the chicken. Am. J. Vet. Res. 39:305–308. Hannun, Y. A., and C. M. Linardic, 1993. Sphingolipid breakdown products: Anti-proliferative and tumor-suppressor lipids. Biochim. Biophys. Acta 1154:223–236. Harrison, L. R., B. M. Colvin, J. T. Greene, L. E. Newman, and R. C. Cole, Jr., 1990. Pulmonary edema and hydrothorax in swine produced by fumonisin B1, a toxic metabolite of Fusarium moniliforme. J. Vet. Diagn. Invest. 2:217–221. Hovi, T., J. Suni, L. Hortling, and A. Vaheri, 1978. Stimulation of chicken lymphocytes by T- and B-cell mitogens. Cell. Immunol. 39:70–78. Huff, W. E., and J. A. Doerr, 1981. Synergism between aflatoxin and ochratoxin A in broiler chickens. Poultry Sci. 60:550–555. Huff, W. E., R. B. Harvey, L. F. Kubena, and G. E. Rottinghaus, 1988. Toxic synergism between aflatoxin and T-2 toxin in broiler chickens. Poultry Sci. 67:1418–1423. Javed, T., G. A. Bennett, J. L. Richard, M. A. Dombrink-Kurtzman, L. M. Cote, and W. B. Buck, 1993. Mortality in broiler chicks on feed amended with Fusarium proliferatum culture material or with purified fumonisin B1 and moniliformin. Mycopathologia 123:171–184. Kubena, L. F., T. S. Edrington, C. Kamps-Holtzapple, R. B. Harvey, M. H. Elissalde, and G. E. Rottinghaus, 1995. Influence of fumonisin B1, present in Fusarium moniliforme culture material, and T-2 toxin on turkey poults. Poultry Sci. 74:306–313. Ledoux, D. R., T. P. Brown, T. S. Weibking, and G. E. Rottinghaus, 1992. Fumonisin toxicity in broiler chicks. J. Vet. Diagn. Invest. 4:330–333. Marasas, W.F.O., T. S. Kellerman, W.C.A. Gelderblom, J.A.W. Coetzer, P. G. Thiel, and J. J. Vander Lugt, 1988. Leukoencephalomalacia in a horse induced by fumonisin B1 isolated from Fusarium moniliforme. Onderstepoort J. Vet. Res. 55:197–203. Marijanovic, D. R., P. Holt, W. P. Norred, C. W. Bacon, K. A. Voss, and P. C. Stancel, 1991. Immunosuppressive effects of Fusarium moniliforme corn cultures in chicks. Poultry Sci. 70:1895–1901. Martinova, E. A., 1996. Fumonisin B1-immunological effects. Pages 331–342 in: Natural Toxins. B. R. Singh and A. T. Tu, ed. Plenum Press, New York, NY. National Research Council, 1994. Nutrient Requirements of Poultry. 8th rev. ed. National Academy Press, Washington, DC. Neldon-Ortiz, D. L., and M. A. Qureshi, 1992. The effects of direct and microsomal activated aflatoxin B1 on chicken peritoneal macrophages in vitro. Vet. Immunol. Immunopathol. 31:61–76. Osweiler, G. D., M. E. Kehrli, J. R. Stabel, J. R. Thurston, P. F. Ross, and T. M. Wilson, 1993. Effects of fumonisin-contaminated corn screenings on growth and health of feeder calves. J. Anim. Sci. 71:459–466. Patten, V. H., S. J. Shin, J. Cole, C. W. Watson, and W. H. Fales, 1995. Evaluation of a commercial automated system and soft-

1282

LI ET AL.

ware for identification of veterinary bacterial isolates. J. Vet. Diagn. Invest. 7:506–508. Pier, A. C., and K. L. Heddleston, 1970. Effect of aflatoxin on immunity in turkeys. I. Impairment of actively acquired resistance to bacterial challenge. Avian Dis. 14:797–809. Pier, A. C., and M. E. Mcloughlin, 1985. Mycotoxic suppression of immunity. Pages 507–519 in: Trichothecenes and Other Mycotoxins. J. Lacey, ed. Wiley and Sons, Chichester, U.K. Qureshi, M. A., J. D. Garlich, W. M. Hagler, Jr., and D. Weinstock, 1995. Fusarium proliferatum culture material alters several production and immune performance parameters in White Leghorn chickens. Immunopharmacol. Immunotoxicol. 17:791– 804. Qureshi, M. A., and W. M. Hagler, Jr., 1992. Effects of fumonisin B1 exposure on chicks macrophage functions in vitro. Poultry Sci. 71:104–112. Rottinghaus, G. E., B. Olsen, and G. D. Osweiler, 1982. Rapid screening method for aflatoxin B1, zearalenone, ochratoxin A, T-2 toxin, diacetoxyscirpenol and vomitoxin. Pages 477– 484 in: Proceedings of the 25th Annual American Association of Veterinary Laboratory Diagnosticians, Nashville, TN. Snedecor, G. W., and W. G. Cochran, 1967. Statistical Methods. The Iowa State University Press, Ames, IA. Sydenham, E. W., P. G. Thiel, W.F.O. Marasas, G. S. Shephard, D. J. VanSchalkwyk, and K. Koch, 1990. Natural occurrence of some Fusarium mycotoxins in corn from low and high esophageal cancer prevalence areas of the Transkei, Southern Africa. J. Agric. Food Chem. 38:1900–1903.

Thaxton, J. P., H. T. Tung, and P. B. Hamilton, 1974. Immunosuppression in chicks by aflatoxin. Poultry Sci. 53:721–725. Wang, E., W. P. Norred, C. W. Bacon, R. T. Riley, and A. H. Merrill, Jr., 1991. Inhibition of sphingolipid biosynthesis by fumonisins. J. Biol. Chem. 266:11486–11490. Weibking, T. S., D. R. Ledoux, A. J. Bermudez, and G. E. Rottinghaus, 1994. Individual and combined effects of feeding Fusarium moniliforme culture material, containing known levels of fumonisin B1, and aflatoxin B1 in the young turkey poult. Poultry Sci. 73:1517–1525. Weibking, T. S., D. R. Ledoux, A. J. Bermudez, J. R. Turk, G. E. Rottinghaus, E. Wang, and A. H. Merrill, Jr., 1993a. Effects of feeding Fusarium moniliforme culture material, containing known levels of fumonisin B1, on the young broiler chick. Poultry Sci. 72:456–466. Weibking, T. S., D. R. Ledoux, A. J. Bermudez, and G. E. Rottinghaus, 1993b. Effects of fumonisin B1 present in Fusarium moniliforme culture material, in turkey poults. Poultry Sci. 72(Suppl. 1):197. (Abstr.) Wu, W., M. E. Cook, and E. B. Smalley, 1991. Decreased immune response and increased incidence of tibial dyschondroplasia caused by fusaria grown in sterile corn. Poultry Sci. 70:293–301. Yoo, H. S., 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-PK1 cells. Toxicol. Appl. Pharmacol. 114:9–15.