Effects of Feeding Fusarium verticillioides (Formerly Fusarium moniliforme) Culture Material Containing Known Levels of Fumonisin B1 in Japanese Quail (Coturnix coturnix japonica) R. K. Asrani,*1 R. C. Katoch,† V. K. Gupta,* S. Deshmukh,* N. Jindal,‡ D. R. Ledoux,§ G. E. Rottinghaus,§ and S. P. Singh* *Department of Veterinary Pathology, †Department of Veterinary Microbiology, Dr. CG Negi College of Veterinary and Animal Sciences, CSK Himachal Pradesh Agricultural University, Palampur—176 062, India; ‡Department of Veterinary Epidemiology and Preventive Medicine, College of Veterinary Sciences, CCS Haryana Agricultural University, Hisar—125 004, India; and §Fusarium/Poultry Research Laboratory, University of Missouri, Columbia 65211 ABSTRACT One hundred fifty 1-d-old quail chicks (Coturnix coturnix japonica) were divided into 2 groups. The 2 groups were designated as controls (CX) and fumonisinfed birds (FX) with each containing 50 and 100 chicks, respectively. The birds in group CX were maintained on quail mash alone, whereas the birds in group FX were maintained on diets supplemented with 300 ppm of fumonisin B1 from Fusarium verticillioides (formerly Fusarium moniliforme) culture material from 1 d. Quail chicks in both groups were examined daily for clinical signs and mortality. Five randomly selected quail from each group were individually weighed on 0, 7, 14, 21, and 28 d postfeeding (DPF). After weighing, blood was collected from these birds at 7, 14, 21, and 28 DPF for hematological studies and at 14, 21, and 28 DPF for biochemical studies. Fumonisin B1-fed birds (FX) had ruffled feathers, reduced feed and water intake, poor body growth, and greenish
mucus diarrhea with 59% mortality. Nearly 30% of the fumonisin B1-fed birds showed nervous signs during the 4-wk experimental period. From 7 DPF onward, BW in group FX were significantly lower than those in group CX. Fumonisin feeding significantly increased hemoglobin, packed cell volume, total erythrocyte count, and total leukocyte count. There was also a significant increase in aspartate transaminase and alanine transaminase in the fumonisin-fed group. Fumonisins significantly increased concentrations of total serum protein and albumin on 14 and 21 DPF, serum calcium and cholesterol levels from 14 DPF onward, and creatinine from 21 DPF onward. This study revealed that the addition of F. verticillioides culture material supplying a level of 300 ppm of FB1/kg of diet is highly toxic to quail chicks, resulting in heavy mortality, decreased growth rate, and significant alterations in hemato-biochemical parameters.
Key words: Fusarium verticillioides culture material, fumonisin B1, Japanese quail, nervous signs, hematology 2006 Poultry Science 85:1129–1135
INTRODUCTION The fumonisins were isolated for the first time from the fungus Fusarium verticillioides (formerly Fusarium moniliforme) in South Africa (Bezuidenhout et al., 1988). A number of fumonisins have since been isolated and characterized, but fumonisin B1 (FB1) remains the most toxic compound (Gelderblom et al., 1992; Seo and Lee, 1999). Fumonisin B1 has been reported to occur naturally in corn and corn screenings at levels as high as 195 and 330 mg of FB1/kg, respectively (Ross et al., 1991). Similar studies carried out by various workers in India have found high levels of FB1 in corn or poultry feeds or both (Shetty and
2006 Poultry Science Association Inc. Received October 18, 2005. Accepted March 3, 2006. 1 Corresponding author:
[email protected]
Bhat, 1997; Jindal et al., 1999). In naturally contaminated corn or corn-based feeds, or in purified form, FB1 has been reported to cause equine leukoencephalomalacia (Marasas et al., 1988), porcine pulmonary edema, and hydrothorax syndrome (Harrison et al., 1990). Fumonisin B1 also causes liver toxicity and liver cancer in rats and atherosclerosis in monkeys (Norred, 1993). Fumonisins have been found to be epidemiologically associated with a high incidence of esophageal cancer in human beings in South Africa and China (Rheedar et al., 1992; Yoshizawa et al., 1994). The majority of reports on the toxic effects of FB1 in avian species pertain to chickens and turkeys, in which it has been found when fed at high levels to be associated with reduced BW and feed intake, diarrhea, poor performance, and alterations in hematological and biochemical parameters with increased activity of the enzymes alanine transaminase and aspartate transaminase (Brown et al., 1992; Ledoux et al., 1992a; Weibking et al., 1993a,b; Ber-
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mudez et al., 1996; Espada et al., 1997; Henry et al., 2000). Unlike aflatoxins and ochratoxins, the susceptibility of quail to FB1 is not well known. In earlier studies we reported the effects of feeding Fusarium verticillioides culture material (FCM) supplying 150 ppm of FB1 to Japanese quail (Deshmukh et al., 2005b,c). The present study was undertaken to determine the effects of feeding FCM containing 300 ppm of FB1 in relation to development of clinical signs, mortality, growth response, and certain hemato-biochemical alterations in Japanese quail.
MATERIALS AND METHODS Experimental Birds, Rearing System, and Diets The present study was conducted with Japanese quail chicks procured from a breeding farm (Central Poultry Breeding Farm, Chandigarh, India). Before housing the quail chicks, the experimental rooms, brooder batteries, and cages were thoroughly cleaned with 2.5% phenol and subsequently fumigated with formaldehyde gas. Quail were kept in cage brooders fitted with electrical bulbs on a 24-h constant light schedule and fed quail mash (Japanese quail ration, Department of Animal Nutrition, COVAS, CSK HPKV, Palampur, India) from d 1 until the end of the experiment. Excreta swabs taken from 15 randomly selected quail on 1 d were inoculated in selenite broth and then incubated for 48 h at 37°C, followed by inoculation on brilliant green agar plates. The excreta samples were negative on bacteriological examination. Feed was autoclaved for 15 min at 15 pounds pressure prior to feeding or mixing with FB1 culture material. Boiled and subsequently cooled water was given to the birds throughout the experiment. Feed and water were given ad libitum, and no medication was given during the entire period of the experiment. Fusarium verticillioides culture material was prepared using Fusarium moniliforme M-1325 (Fusarium Research Center, Pennsylvania State University, University Park, PA; MRC 826; now renamed Fusarium verticillioides) per the method described by Weibking et al. (1993a). Dietary treatment containing FB1 was prepared by incorporating ground FCM that contained 6,000 mg of FB1/kg of culture material to the quail mash. It was mixed thoroughly in the feed to provide the desired level of 300 ppm of FB1, and the culture material made up 5% of the ration. The FCM was not incorporated into the control group feed. Diets were screened before being fed to the birds and found to be free of the following mycotoxins: aflatoxin B1 and ochratoxin A (Scott, 1995) and fumonisins (control diet; Thiel et al., 1993). Though the F. verticillioides culture material used in the present study contained known levels of FB1, however, the possibility of presence of other less toxic fumonisin metabolites as well as unknown toxins could not be ruled out. The animal care and experimental protocols were duly approved by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA).
Experimental Design and Sample Collection One hundred fifty 1-d-old quail were randomly divided into 2 groups (CX and FX with 50 and 100 chicks, respectively). The birds of both the groups were housed in the same room. The number of quail chicks in the FX group was higher because of the expected mortality due to fumonisins. The birds in group CX were maintained on quail mash alone, whereas in group FX the diets were supplemented with FCM that supplied 300 ppm of FB1 from 1 d of age. Quail chicks in both groups postfeeding were examined daily for clinical signs and mortality. Five quail chicks from each group were randomly selected and weighed at 0, 7, 14, 21, and 28 d postfeeding (DPF). After weighing, 1 to 3 mL of blood was collected via cardiac puncture from these birds at 7, 14, 21, and 28 DPF for hematologic determinations and at 14, 21, and 28 DPF for serum biochemistry. For hematology, blood was collected in EDTA (2 mg/mL) tubes, whereas for biochemical studies blood was collected without anticoagulant. The birds were killed after collection of blood samples at each of the aforementioned intervals.
Hematology and Serum Biochemistry Hematological parameters included determination of hemoglobin (Hb) concentration by the cynamethemoglobin method using Drabkin’s solution (Makarem, 1974), packed cell volume (PCV) by the microhematocrit method (Cohen, 1967), and total erythrocyte count (TEC) and total leukocyte count (TLC) by the method of Natt and Herrick (1952). Alanine transaminase, aspartate transaminase, total serum proteins (TSP), albumin, cholesterol, creatinine, and calcium were determined using diagnostic kits (Transasia Biomedicals Ltd., Mumbai, India) according to the manufacturer’s recommendations in a fully automatic Blood Chemistry Analyzer (RA 50 Autochemistry System).
Bacteriological Isolations Sections of liver tissue from a few dead birds were subjected to bacteriological isolations in selenite broth and brilliant green agar plates.
Statistical Analysis The mean of control (CX) and treated group (FX) were compared using t-test at 95% confidence level (P < 0.05) for each parameter studied. The data was also analyzed by Duncan’s Multiple Range test (2-way ANOVA) to determine the effect of FB1 treatment, age, and their interaction (Snedecor and Cochran, 1967).
RESULTS AND DISCUSSION None of the CX birds exhibited any apparent adverse signs or symptoms during the course of the experiment. In
FUMONISIN B1 TOXICITY IN JAPANESE QUAIL
Figure 1. Downward bending of head and neck in Japanese quail kept on diet containing Fusarium verticillioides culture material.
general, throughout the experiment, the FX birds revealed symptoms of ruffled feathers, reduced feed and water intake, poor body growth, and greenish mucus diarrhea. Diarrhea was observed from the 5 d of FB1 feeding when less than 10% of the birds were seen passing slight watery mucoid discharge in the droppings. Subsequently, from 10 DPF onward, the percentage of birds exhibiting diarrhea increased rapidly and was evident from the presence of mucus in the droppings during cleaning operations of the cage floor. Diarrhea was found to be more severe on 14 DPF and onward. The droppings frequently had a bright greenish tinge. Because of diarrhea in chicks of the FX group, the birds also appeared dull and depressed. The clinical signs of lowering the head and sometimes tucking it up into the thoracic cavity along with backward and circling movements were observed in only 3 birds at 11 DPF in the FX group, but by the end of the experiment, nearly 30% of the birds had shown these symptoms (Figure 1). Less than 5% of the birds showed signs of leg weakness during the entire period of the experiment. However, the signs of leg weakness were prominent in the studies conducted by Kohler et al. (1978), who reported that feeding of Fusarium moniliforme culture material resulted in malabsorption of calcium and vitamin D3 from the intestine and the leg weakness/lameness simulated rickets. The findings of the present study were consistent with those of Brown et al. (1992) and Prathap Kumar et al. (1997). The clinical and pathological response to FB1 toxicity has been found to be different in various animal and avian species. Japanese quail is the only avian species that has shown some nervous symptoms, and development of these symptoms has been found to be dose-dependent. In our earlier studies in which FCM supplying 150 ppm of FB1/kg of feed was given from 5 d of age, none of the quail chicks showed nervous symptoms even up to 37 d post-FB1 feeding (Deshmukh et al., 2005b). In another study in which FCM supplying 325 ppm of FB1/kg of
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feed was fed to quail chicks from 7 d of age, nervous symptoms were observed in nearly 21% of the birds and appeared as early as 10 DPF (R. K. Asrani, personnel communication). Clinical FB1 toxicity in broiler chickens and turkeys has been found to be primarily associated with reduced body weight gain and diarrhea (Ledoux et al., 1992a). Whereas diarrhea in broilers and turkeys has been found to occur when the level of FCM exceeds 10% of the diet, quail have been found to be sensitive to levels as low as 2.5% FCM, and a level of 5% FCM in the present study led to severe diarrhea in the quail in addition to nervous signs. Japanese quail are the only species other than equines that have shown nervous signs. The disease equine leukoencephalomalacia due to FB1 toxicity is clinically characterized by acute deaths with or without neurological symptoms of ataxia, head pressing, and circling movements of 1 to 2 d duration (Wilson et al., 1990). On the other hand, the clinical disease in pigs consists of acute onset of dyspnea followed by weakness, cyanosis, and death due to severe pulmonary edema and hydrothorax (Diaz and Boermans, 1994). Nervous signs observed in the present study may be attributed to blockage of sphingolipid synthesis by the fumonisins since sphingolipids predominate in the central and peripheral nervous system mainly as the myelin lipid (Wang et al., 1991). No mortality was observed in quail chicks in the CX group throughout the experiment. However, mortality in chicks in the FX group started as early as 8 DPF. There was mortality of 3 to 5 birds almost daily up to 24 DPF in this group. A total of 59 (59.0%) quail chicks died during the 4-wk experimental period. Fumonisin B1 was confirmed as the cause of mortality on the basis of histopathological studies and by absence of any bacterial growth on cultural examination. In contrast to broiler chicks (Brown et al., 1992) and turkey poults (Ledoux et al., 1992b), mortality was higher in fumonisin-fed quail chicks in the present study. This may be due to a higher susceptibility of quail to the toxic effects of fumonisins compared with broiler chickens and turkey poults. A significant increase in BW of quail chicks was observed with advancing age. The BW of quail chicks in the FX group also increased with age; however, the BW in this group were significantly lower than chicks in the CX group from 7 DPF onward (Table 1). By 21 DPF, BW in group FX was only 54% of group CX, and at 28 DPF, the BW of the FX group was only 35% of group CX. Mean treatment effect at the end of the experiment revealed a significant decrease in BW of quail chicks on fumonisin feeding. The interaction between FB1 treatment and age of quail chicks was also significant (Table 1). Decreased BW in broiler chickens and turkey poults fed FB1-contaminated diets have also been previously reported (Brown et al., 1992; Prathap Kumar et al., 1997). Piramanayagam and George (2002) reported that feeding of graded doses of FB1 (80, 160, and 320 ppm) decreased BW and feed intake of broiler chicks. Ogido et al. (2004) reported that feeding 10 ppm of FB1 to laying Japanese quail chicks also reduced the BW and feed intake. The decrease in BW in avian species may be due to the irritating effects
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Table 1. Effects of feeding fumonisin B1, supplied by Fusarium verticillioides (formerly F. moniliforme) culture material, on BW and hematology of Japanese quail1 Days postfeeding Parameter
Group
7
14
21
28
CX FX Mean age effect4
15.00 ± 0.87a 9.72 ± 1.19b 12.36 ± 1.18d
23.61 ± 2.41a 16.73 ± 2.23b 20.17 ± 2.32c
53.08 ± 3.06a 28.96 ± 5.10b 41.02 ± 4.90b
72.77 ± 1.59a 46.82 ± 3.54b 59.79 ± 3.21a
41.00 ± 4.96a 26.34 ± 3.27b
CX FX Mean age effect
4.64 ± 0.27a 4.46 ± 0.31a 4.55 ± 0.19b
5.60 ± 0.17a 5.58 ± 0.20a 5.59 ± 0.12a
5.22 ± 0.25b 6.38 ± 0.58a 5.80 ± 0.35a
5.91 ± 0.10b 6.12 ± 0.21a 6.01 ± 0.11a
5.45 ± 0.13a 5.73 ± 0.20a
CX FX Mean age effect
b
33.40 ± 1.69 30.60 ± 1.63a 32.00 ± 1.20b
a
36.80 ± 1.71 37.40 ± 0.40a 37.10 ± 0.83a
b
32.60 ± 2.35 41.40 ± 3.73a 37.00 ± 2.54a
b
36.60 ± 1.49 38.80 ± 0.99a 37.70 ± 0.91a
35.20 ± 0.92 37.40 ± 1.12a
CX FX Mean age effect
a
4.88 ± 0.24 4.69 ± 0.25a 4.78 ± 0.17a
a
3.48 ± 0.55 3.51 ± 0.25a 3.50 ± 0.28b
b
4.24 ± 0.49 4.75 ± 0.46a 4.49 ± 0.33a
b
4.54 ± 0.27 5.21 ± 0.21a 4.87 ± 0.22a
4.33 ± 0.20 4.67 ± 0.18a
CX FX Mean age effect
11.80 ± 0.86 17.40 ± 2.23a 14.60 ± 1.46b
9.40 ± 1.12 16.20 ± 3.05a 12.80 ± 1.90b
16.60 ± 4.04 25.40 ± 2.92a 21.00 ± 2.77a
19.20 ± 2.34 24.60 ± 2.42a 21.00 ± 1.83a
15.24 ± 1.44 20.90 ± 1.55a
Mean BW (g)
Hb (gm%)
PCV (%)
TEC (106/mm3)
TLC (103/mm3)
Age × treatment effect3
Mean treatment effect2
b
b
b
b
9.56 (S)
2.09 (NS) a
2.91 (NS) b
0.75 (NS) b
1.11 (NS)
Values within columns (between groups CX and FX) with different superscripts for a parameter are significantly different by t-test (P ≤ 0.05). All the values are mean ± SE of 5 quail chicks at each interval. Hb = hemoglobin content; PCV = packed cell volume; TEC = total erythrocyte count; and TLC = total leukocyte count. CX = birds fed quail mash alone; and FX = birds fed quail mash supplemented with Fusarium verticillioides culture material. 2 Values within a column showing mean treatment effect with different superscripts for a parameter are significantly different by ANOVA (P ≤ 0.05). 3 F value for a parameter indicating interaction between FB1 treatment and age of quail chicks (S = significant) by ANOVA (P ≤ 0.05). 4 Values within a row showing mean age effect with different superscripts for a parameter are significantly different by ANOVA (P ≤ 0.05). a,b 1
of fumonisin B1 on the gut mucosa or induced alteration of normal intestinal flora resulting in malabsorption of important nutrients (Brown et al., 1992) or FCM-induced feed refusal (Bermudez et al., 1996). Values of erythrocytic indices (Hb, PCV, and TEC), in general, showed an increase with progression of age in both experimental groups, and the mean values of all the 3 indices showed more or less a similar trend at all intervals. No major difference between the groups was observed in overall mean values for Hb and PCV, although the values appeared significantly higher (t-test) in quail chicks in group FX compared with chicks fed quail mash alone (group CX) from 21 DPF onward (Table 1). The overall mean values of TEC showed a significant increase (ANOVA) on fumonisin feeding. However, FB1 treatment × age interaction for these erythrocytic indices was nonsignificant (Table 1). Earlier studies on broiler chicks and turkey poults have shown only a few significant changes or differences in Hb, PCV, and TEC following FB1 treatment (Ledoux et al., 1992a; Weibking et al., 1993a,b). In the present study, the increase in erythrocytic indices may have been the result of relative polycythemia in proportion to water lost by diarrhea. Total leukocyte count values showed an increasing trend with age in both groups, and effect of age was evident from 21 d onward when mean TLC values were significantly higher than the values at 7 and 14 d. The mean TLC values were significantly higher (t-test) in the FX group than in the CX group from 7 DPF till the end of the experiment (Table 1). Overall treatment effect showed a significantly higher
mean value of TLC in the FX group compared with CX; however, FB1 treatment × age interaction for this parameter was nonsignificant (Table 1). Significantly higher values of TLC in the FB1-fed quail chicks is consistent with the findings of Ledoux et al. (1996) and Bermudez et al. (1996), who also recorded higher counts of total leukocytes on feeding fumonisin-contaminated diets to turkey poults. Though differential leukocyte count was not carried out in the present study, the increase in total leukocyte count in fumonisin-fed quail chicks may have been due to an increase in the percentage of heterophils due to the presence of degenerative and necrotic lesions in the liver as also suggested by Brown et al. (1992) and Weibking et al. (1993a). There were not many variations in the biochemical parameters in chicks fed quail mash alone or with FCM with progression of age as evident from the overall mean values showing the age effect (Table 2). However, significant changes in all the parameters except for serum albumin and TSP were recorded on fumonisin feeding, which is evident from overall mean values showing the treatment effect (Table 2). The interaction between FB1 treatment and the advancing age for all the biochemical parameters studied (except for TSP) was nonsignificant (Table 2). Feeding FB1 (group FX) induced a significant increase in alanine transaminase and aspartate transaminase activities from 14 DPF onward (Table 2) in comparison to group CX. The increase in enzyme activities may be due to damage or necrosis of the hepatocytes (Benjamin, 1985; Ledoux et al., 1992a; Weibking et al., 1993a,b;
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Table 2. Effects of feeding fumonisin B1, supplied by Fusarium verticillioides (formerly F. moniliforme) culture material, on certain biochemical parameters of Japanese quail1 Days postfeeding (DPF) Parameter Alanine transaminase (IU/L) Aspartate transaminase (IU/L) Albumin (g/dL) Total serum proteins (g/dL) Cholesterol (mg/dL) Creatinine (mg/dL) Calcium (mg/dL)
Group
14
CX FX Mean age effect4 CX FX Mean age effect CX FX Mean age effect CX FX Mean age effect CX FX Mean age effect CX FX Mean age effect CX FX Mean age effect
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
14.00 21.00 17.50 200.60 330.80 265.70 0.94 1.16 1.05 3.10 4.26 3.68 153.00 252.20 202.60 1.18 1.16 1.17 8.86 13.02 10.94
21 b
1.87 2.02a 1.74a 28.79b 27.25a 28.63a 0.07b 0.12a 0.07a 0.21b 0.20a 0.23a 4.88b 17.24a 18.56a 0.06a 0.01a 0.02a 0.41b 0.87a 0.82a
13.80 17.80 15.80 222.80 278.60 250.20 0.90 1.00 0.95 3.40 3.96 3.68 145.40 236.40 190.90 1.22 1.17 1.19 9.82 11.14 10.48
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
Mean treatment effect2
28 b
3.07 2.18a 1.89a 7.92b 38.11a 20.57a 0.05b 0.08a 0.05b 0.38b 0.11a 0.21a 4.62b 14.65a 16.80a 0.02a 0.03b 0.02a 1.09b 0.69a 0.65a
12.50 18.90 15.70 206.10 311.60 258.85 1.15 1.14 1.14 4.02 3.84 3.96 151.90 225.50 188.70 1.20 1.09 1.15 8.78 9.94 9.16
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.60b 0.97a 0.92a 7.18b 17.54a 15.21a 0.05a 0.06a 0.04a 0.20a 0.44a 0.18a 9.21b 11.21a 11.01a 0.02a 0.01b 0.01a 0.71b 1.86a 0.75b
Age × treatment effect3
13.20 ± 0.89b 19.15 ± 0.87a 208.90 ± 7.92b 308.15 ± 14.38a 1.03 ± 0.04a 1.11 ± 0.05a 3.63 ± 0.16a 4.02 ± 0.16a 150.55 ± 4.79b 234.90 ± 7.93a 1.20 ± 0.01a 1.13 ± 0.01b 9.06 ± 0.45b 11.36 ± 0.75a
0.41 (NS) 1.32 (NS) 1.26 (NS) 3.47 (S) 0.70 (NS) 1.50 (NS) 0.56 (NS)
Values within columns (between groups CX and FX) with different superscripts for a parameter are significantly different by t-test (P ≤ 0.05). All the values are mean ± SE of 5 quail chicks at each interval. CX = birds fed quail mash alone; and FX = birds fed quail mash supplemented with Fusarium verticillioides culture material. 2 Values within a column showing mean treatment effect with different superscripts for a parameter are significantly different by ANOVA (P ≤ 0.05). 3 F value for a parameter indicating interaction between FB1 treatment and age of quail chicks (S = significant) by ANOVA (P ≤ 0.05). 4 Values within a row showing mean age effect with different superscripts for a parameter are significantly different by ANOVA (P ≤ 0.05). a,b 1
Satheesh et al., 2004; Jindal et al., 2005). The TSP contents increased in CX group with progression of age, in contrast to a declining trend in FX group. Fumonisin treatment significantly increased the concentrations of TSP and albumin in comparison with controls on 14 and 21 DPF; however, overall mean treatment effect across age of the birds between FX and CX groups was not significant (Table 2). Increased levels of albumin and TSP along with a concurrent increase in globulin concentration have been attributed to dehydration (Duncan et al., 1994) or relative polycythemia in proportion to water lost due to diarrhea (Benjamin, 1985). In contrast, Weibking et al. (1993b) and Espada et al. (1997) reported a decrease in albumin and TSP in broiler chicks fed FB1 throughout the experiment. There was a significant increase in serum calcium and cholesterol levels in chicks fed FB1 from 14 DPF until the end of the experiment (Table 2). Increases in levels of calcium and cholesterol in FB1-fed broiler chicks had been reported earlier (Ledoux et al., 1992a). In the present study, increase in serum calcium levels may be linked to disruption of sphingolipid metabolism by fumonisins as long chain sphingoid bases have been shown to induce release of Ca2+ from intracellular stores (Merrill et al., 1996) sequestered in mitochondria and endoplasmic reticulum (Cotran et al., 1994). In a previous study, the major toxic effects of FB1 in quail chicks were observed in the rough endoplasmic reticulum and mitochondria (Deshmukh et al., 2005c). Increase mobilization of calcium from internal stores (Shatrov et al., 1997) may not only open the cell surface calcium channel (van Koppen et al., 1996),
but cell membrane damage through activation of phospholipases or activation or inhibition of other enzymes of lipid signaling pathways in the presence of increase levels of cytosolic free calcium may have been associated with movement of calcium to extracellular space in exchange for sodium. The increase in cholesterol level from feeding FB1 may be due to hepatocellular damage (Benjamin, 1985) and is also supported by the necropsy finding of a pale discoloration of the liver. However, Weibking et al. (1993b) observed decreased levels of serum cholesterol and alkaline phosphatase on feeding fumonisin-contaminated diets to turkey poults. Serum levels of creatinine were significantly reduced in birds fed FB1 (FX group) from 21 DPF; however, a small decrease was also observed earlier at 14 DPF (Table 2). A possible contributing factor to the decrease in creatinine may be liver damage because the liver is responsible for methylation of glycocyamine by active methionine, which synthesizes creatine first prior to the synthesis of creatinine, which is formed largely in muscles by irreversible nonenzymatic dehydration of creatine phosphate (Rodwell, 1988). Another factor possibly contributing to the decreased creatinine levels may be the loss of cross striations, thinning of cardiomyocytes, and muscle necrosis by FB1 (Ledoux et al., 1996; Deshmukh et al., 2005a). Thus, the present study revealed that feeding fumonisin B1 from F. verticillioides culture material to quail chicks can produce nervous signs, diarrhea, high mortality, adverse effects on BW and feed intake, and alterations in hemato-biochemical parameters. Although the effects on
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growth and hemato-biochemical parameters in quail chicks were not different from those reported earlier in broiler chickens and turkey poults, the severity was more pronounced, thereby suggesting FB1 may be a concern to the quail industry. Fumonisins act by altering the sphingolipids metabolism because the polyhydric alcohol moiety of fumonisin resembles the complex amino alcohol sphingosine, which is the backbone of sphingolipids. Furthermore, free sphinganine is growth inhibitory and cytotoxic for cells. The accumulation of sphinganine has been considered to be the cause of pathological alterations in FB1 toxicosis in animals (Merrill et al., 1997). In India, corn is the major ingredient of poultry feed (including quail) and makes up 50 to 60% of the ration. In the Haryana state of India, 91% corn samples and 84% of poultry feed samples were found to contain varying levels of FB1 (Jindal et al., 1999). Chatterjee and Mukherjee (1994) earlier reported levels of 300 to 366 ppm of FB1 in the Fusarium-infested corn samples. These levels of FB1 may cause toxicity in quail chicks. Furthermore, fumonisin B1 has also been shown to be immunosuppressive; it may predispose quail chicks to other secondary infections, thereby increasing the chances of mortality or decreased production. Although no studies have been reported on the immunosuppressive effects of FB1 in quail, the results of a previous study indicated that depressive effects on growth and mortality were higher in FB1-fed quail chicks infected with Salmonella Gallinarum than in chicks fed FB1 or infected with S. Gallinarum alone (Deshmukh et al., 2005b). In addition, the concurrent presence of other mycotoxins like aflatoxins, ochratoxin, T-2 toxin, etc., in feed may act synergistically or additively causing more severe effects than the individual toxin. In Southern India, cooccurrence of fumonisin B1 and aflatoxins was recorded in feed samples (Shetty and Bhat, 1997). Prathap Kumar et al. (1997) recorded diarrhea, decreased BW and feed intake, drop in egg production, and mortality at 2 layer farms in Andhra Pradesh, India, due to the concurrent presence of fumonisins and aflatoxins in feed, suggesting that regular screening of poultry feed samples/ ingredients for mycotoxins may avert economic losses.
ACKNOWLEDGMENTS Authors are grateful to S. K. Sharma, Associate Professor, Department of Veterinary Surgery and Radiology, CSK Himachal Pradesh Agricultural University, Palampur, for his assistance in the completion of this study.
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