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Individual and Combined Effects of Fumonisin B₁ and Moniliformin on Clinicopathological and Cell-Mediated Immune Response in Japanese Quail

D. Sharma^*, R. K. Asrani^*,1, D. R. Ledoux, N. Jindal, G. E. Rottinghaus and V. K. Gupta^*

^* Department of Veterinary Pathology, Dr. G. C. Negi College of Veterinary and Animal Sciences, CSK Himachal Pradesh Agricultural University, Palampur–176062, India; Fusarium/Poultry Research Laboratory, University of Missouri, Columbia 65211; and Department of Veterinary Epidemiology and Preventive Medicine, College of Veterinary Sciences, CCS Haryana Agricultural University, Hisar-125 004, India

¹ Corresponding author: asranirk{at}gmail.com

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
A total of 390 one-day-old quail chicks (Coturnix coturnix japonica) were divided into 4 groups (3 replicates per treatment), viz. CX, FX, MX, and FM, containing 75, 105, 105, and 105 birds, respectively. Birds in the control group (CX) were fed quail mash alone, whereas birds in group FX were fed 200 ppm of fumonisin B₁ (FB₁) from Fusarium verticillioides culture material; group MX was fed 100 ppm of moniliformin (M) from Fusarium fujikuroi culture material; and group FM was fed a combination of 200 ppm of FB₁ and 100 ppm of M. Diets were fed from d 1 to 35 to study clinical signs, growth response, serum biochemical changes, and cell-mediated immune response. Birds fed FB₁ (FX) showed ruffled feathers and poor growth. Birds in group MX appeared more stunted than those in group FX and exhibited signs of poor feathering and decreased feed and water intake. Clinical signs observed in group FM were more or less similar to those observed in groups FX and MX. Total mortality was 12.38, 7.62, and 20.95% for groups FX, MX, and FM, respectively. Mean BW in groups FX, MX, and FM were significantly lower than those in the control group (CX) at almost all intervals. Total serum proteins, albumin, cholesterol, aspartate transaminase, lactate dehydrogenase, and creatine kinase values were higher in all treatment groups compared with the control group. Cell-mediated immune response was more or less comparable in groups CX and MX, whereas the presence of FB₁ in the diet of groups FX and FM was found to be associated with a gradual increase in skin thickness, and the mononuclear inflammatory cell response was poor as compared with groups CX and MX throughout the study. Except for mortality (additive effect) and serum aspartate transaminase values (less than an additive effect up to 14 DPF), no additive or synergistic effects were observed for any of the other response variables measured in the current study, where all statistical differences were attributed to either one mycotoxin or the other.

Key Words: Fusarium verticillioides culture material • Fusarium fujikuroi culture material • fumonisin B₁ • moniliformin • Japanese quail

	INTRODUCTION

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Fumonisin B₁ (FB₁), a water-soluble Fusarium metabolite, is the most abundant member of the recently discovered group of mycotoxins called fumonisins (Bezuidenhout et al., 1988). The fumonisins were isolated for the first time from the fungus Fusarium moniliforme (now renamed Fusarium verticillioides) in South Africa. A number of fumonisins have since been isolated and characterized, but FB₁ remains the most toxic compound (Gelderblom et al., 1991). 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 researchers in India have also found high levels of FB₁ in maize, poultry feeds, or both (Shetty and Bhat, 1997; Jindal et al., 1999).

Fumonisin B₁, either in naturally contaminated maize or maize-based feeds or in purified form, 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 South Africa and China (Rheedar et al., 1992; Yoshizawa et al., 1994). In chicks, FB₁ has been associated with poor performance, elevated free sphinganine:sphingosine ratios, increased organ weights, decreased immune responses, and organ lesions (Ledoux et al., 1992; Qureshi and Hagler, 1992; Javed et al., 1993; Weibking et al., 1993a; Li et al., 1997). In quail, FB₁ has been reported to cause ruffled feathers, reduced feed and water intake, poor body growth, and greenish mucus diarrhea with 59% mortality (Asrani et al., 2006).

Moniliformin (M) is a water-soluble fungal metabolite produced by several Fusarium species. Moniliformin appears to be a growth regulator in plants and is phytotoxic in corn and tobacco (Vesonder and Golinski, 1989). Moniliformin is highly toxic and results in rapid death in chicks and rats. Although M was discovered more than 2 decades ago (Cole et al., 1973), active investigations on the toxic effects of M in poultry have been initiated only in recent years. Cardiac injury, with alterations in the cardiac electrical conductance, was shown to be a primary cause of mortality in birds (Nagaraj et al., 1996; Reams et al., 1997). Prolonged feeding of M to birds has been shown to cause poor growth performance, increased serum pyruvate levels, and cardiopathy (Ledoux et al., 1993; Morris et al., 1997; Reams et al., 1997). Acute mortality and gross lesions, including ascites, hydropericardium, and myocardial pallor, have been observed in broilers, turkeys, and ducklings (Engelhardt et al., 1989).

Although most research has concentrated on the individual effects of FB₁ and M in poultry, these mycotoxins are often present simultaneously in moldy corn samples (Sydenham et al., 1990). It has been shown that the toxicity of some individual mycotoxins can be increased or decreased when they are present as cocontaminants in feed (Huff et al., 1988; Kubena et al., 1988, 1995). Fumonisin B₁ and M fed in combination to chicks resulted in an additive negative effect on performance (Javed et al., 1993), whereas in turkey poults, no synergistic or additive effects were reported for any parameter measured (Bermudez et al., 1997). Poults fed a combination of FB₁ and M had lower primary and secondary antibody responses and lower relative thymus weights (Li et al., 2000b). Both renal and cardiac lesions were observed in chickens fed a combination of FB₁ and M (Ledoux et al., 2003), but the interactive effects of FB₁ of M were not synergistic and were less than additive in nature. Quail farming is increasing in certain parts of India and corn is frequently used up to 50 to 60% in poultry rations (including quail). In one of our earlier studies, the disease process in quail after FB₁ feeding was shown to be different from that of chickens and turkeys, as characterized by signs of nervousness (Asrani et al., 2006). The susceptibility of quail to M is not well known. Hence, in the present study the individual and combined effects of FB₁ and M were evaluated in Japanese quail.

	MATERIALS AND METHODS

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Cleaning of Experimental Room and Cages

Before the arrival of unvaccinated 1-d-old Japanese quail (Coturnix coturnix japonica) in the department, the experimental room, cages, trays, and cage stands were first washed with tap water and then thoroughly cleaned with 2.5% phenol. This cleaning process was repeated 3 times at intervals of 10 to 15 d. After drying in the sunlight, the cages, trays, and stands were flamed with dry heat by using a blowtorch. The waterers and feeders were thoroughly washed with potassium permanganate solution and sun dried. After placing the cages and other accessories in their designated areas in the room, the room was fumigated with formaldehyde gas just 2 d before arrival of the quail chicks. The cages were cleaned daily with 2.5% phenol until the end of the experiment.

Experimental Birds

The present study was conducted on 390 one-day-old Japanese quail chicks procured from the Central Poultry Development Organization (Chandigarh, India). The quail chicks used in the present study were from the same breeding flock. Excreta swabs taken on d 1 from 20 randomly selected chicks, before dividing them into various groups, were streaked on brilliant green agar plates and examined for bacterial growth after 48 h of incubation at 37°C. The excreta samples were found to be negative for Salmonella on bacteriological examination. The birds were kept under strict hygienic conditions in departmental animal housing throughout the period of the experiment. The animal care and experimental protocol were approved by the university and by the Committee for the Purpose of Control and Supervision of Experiments on Animals.

Feeding Schedule

The quail chicks were maintained on chick mash (quail mash procured from Him Vet Feed Industry, Palampur, India) from d 1 until the end of the experiment. Feed was autoclaved for 15 min at 15 pounds per square inch before feeding or mixing with Fusarium culture material(s). 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. Before feeding, representative samples of chick mash were submitted to the Animal Feed Analytical and Quality Control Laboratory, Veterinary Hospital Campus (Namakkal, Tamil Nadu, India), and to the Department of Veterinary Epidemiology and Preventive Medicine, College of Veterinary Sciences, CCS Haryana Agricultural University (Hisar, Haryana, India), for analysis of common mycotoxins. The feed samples were found to contain aflatoxins at permissible limits (6 µg/kg) and were free of citrinin, FB₁, M, ochratoxin A, T-2 toxin, and zearalenone. The mycotoxins for the present study, FB₁ and M, were supplied by F. verticillioides M-1325 culture material (FCM) and Fusarium fujikuroi M-1214 culture material (MCM), respectively (courtesy of G. E. Rottinghaus, University of Missouri, Columbia, MO). Fusarium verticillioides M-1325 culture material containing 6,200 mg of FB₁/ kg and MCM containing 10,000 mg of M/kg was incorporated at the rate of 3.25 and 1% into the chick mash to supply 200 mg of FB₁/kg of feed and 100 mg of M/kg of feed, respectively. The FCM and MCM were not incorporated into the feed of the control group.

Experimental Design

A total of 390 one-day-old Japanese quail chicks were randomly divided into 4 groups, fumonisin B₁ alone (FX), moniliformin alone (MX), fumonisin B₁ and moniliformin (FM), and chick mash alone (CX), with 105 birds in each group for groups FX, MX, and FM, and with 75 birds in group CX. The present study was conducted with 3 pen replicates of 35 quail per pen in each of the 3 groups FX, MX, and FM, and 3 pen replicates of 25 quail per pen in group CX. Although the duration of the experiment was up to 35 d postfeeding (DPF), clinical signs, growth response, and serum biochemical changes were studied only up to 28 DPF because it was thought that the application of 1-chloro-2,4-dinitrobenzene (DNCB) for the contact hypersensitivity test at 28 DPF and onward might interfere with some clinical parameters. The various dietary treatments, starting from d 1 until 35 d in different groups, are presented in Table 1.

The birds in all the groups were closely observed, at least 3 times daily, for the development of clinical signs and mortality. To see the individual and combined effect of FB₁ and M on BW, 9 randomly selected quail from each treatment group (3 quail per replicate) were weighed at 0, 7, 14, 21, and 28 DPF. After weighing, 2 to 3 mL of blood was collected (at 7, 14, 21, and 28 DPF) via cardiac puncture from each of these birds for determination of serum aspartate transaminase (AST), alanine transaminase (ALT), total serum proteins (TSP), albumin, cholesterol, and creatinine. Determination of lactate dehydrogenase (LDH) and creatine kinase (CK) was done only at 28 DPF. All the biochemical determinations were done by using diagnostic kits (Bayer Diagnostics India Ltd., Baroda, India) in a fully automated Blood Chemistry Analyzer (RA-50 Auto Chemistry System, Bayer Diagnostics) according to the manufacturer’s recommendations. The birds were killed after collection of blood samples at each of the aforementioned intervals.

Contact Hypersensitivity Test

Thirty-six birds from each group (CX, FX, MX, and FM) were subjected to contact hypersensitivity testing, which was assessed by skin hypersensitivity reaction to DNCB. At 28 DPF, birds were first sensitized with DNCB (SRL Sisco Research Lab., Mumbai, India) and subsequently challenged 1 wk later (i.e., at 35 DPF). During this time, birds were continued on their respective dietary treatments. Birds were sensitized by painting 100 µL of 2.5% DNCB dissolved in acetone (4:1) vehicle on one side of the abdominal skin. The challenge was performed by applying 20 µL of 1% DNCB on the other side of the abdominal skin 7 d after sensitization. Before challenge, skin thickness was measured in individual birds from each of the treatment groups. After challenge, the degree of contact hypersensitivity was determined by measuring the change in thickness of the skin caused by swelling. Skin thickness was measured at 1, 2, 3, 6, 12, 24, 48, and 72 h after challenge by using a vernier caliper (Hauptner, Solingen, Germany) on 9 birds from each group (3 quail per replicate). After measuring the skin thickness, 3 birds from each of the treatment groups (CX, FX, MX, and FM; 1 quail per replicate) were euthanized by cervical dislocation at 0 (before challenge), 1, 2, 3, 6, 12, 24, 48, and 72 h after challenge, and pieces of skin tissue were collected in 10% neutral-buffered formalin for histological studies. The percentage increase in skin thickness was calculated by using the following formula to indicate the degree of swelling:

Bacteriological Isolations

Sections of liver tissue from a few dead and killed birds from each of the treatment and control groups at different intervals were subjected to bacteriological isolations in selenite broth and brilliant green agar plates.

Statistical Analysis

Data were subjected to statistical analysis for drawing inferences by using a standard procedure (Snedecor and Cochran, 1967). Treatment means were compared by using Duncan’s multiple range test (2-way ANOVA) to determine the effect of treatment, age, and their interactions (Duncan, 1955). All levels of significance were based on the 95% level of probability.

	RESULTS

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Clinical Signs

None of the birds from the CX group exhibited any clinical signs throughout the course of the experiment. In group FX, clinical signs were detected as early as 3 d after feeding of FB₁ began, when nearly 5% of the birds were seen passing dark tan-colored semisolid excreta. Occasionally, droppings revealed mild to moderate levels of mucus. Subsequently, the percentage of birds exhibiting diarrhea increased rapidly, and by 2 wk post-FCM feeding, a large number of birds had shown increased mucus contents in the droppings during cleaning operations of the cage floor. Although the number of birds exhibiting mucus diarrhea reduced thereafter, the excreta continued to have a pasty consistency until the end of the experiment. In addition, clinical signs of ruffled feathers and stunted growth were observed in almost all birds from 1 wk onward. Occasionally, birds were found sitting on their hocks. In general, the clinical signs in group MX, discernible as early as 2 d post-MCM feeding, were similar to those observed in group FX, except that the diarrhea in group MX revealed a more watery consistency and was observed in a majority of birds up to the second week, and mucus was occasionally seen in the droppings. The diarrheic excreta also appeared whitish in color. The number of diarrheic birds reduced significantly thereafter but fluffy, cotton-like, white, mild pasty droppings, which appeared to be predominantly urates, were recorded from the third week onward. Clinical signs of poor body growth, ruffled feathers, and low feed and water intake were observed consistently throughout the experiment in this group. Although feed consumption was not measured in the present study, daily observations revealed less feed intake by birds fed MCM, FCM, or both. Droopiness of wings was observed in nearly 10% of the birds. The birds in group FM revealed signs more or less similar to those observed in groups FX and MX. Although there was no significant difference in the severity of diarrhea when compared with other groups (FX and MX), feed refusal was comparatively higher in this group, as detected during cleaning of cage floors. Diarrhea was recorded as early as 2 d post-FCM and post-MCM feeding, when a few birds (nearly 5%) were seen passing slightly loose excreta, which appeared dark in color. From d 4 onward, droppings appeared watery, with increased mucus content. Subsequently, after 2 wk, excreta appeared to have a semisolid consistency, with slight mucus seen occasionally and, in some cases, with a whitish tinge. On d 4, a typical posture of resting against the sternum and raising the posterior was seen in some birds, but this was not a consistent feature. The clinical signs of hyperexcitation and ataxia with backward motion to gain balance were observed in only 2 birds in the second week.

Mortality

No mortality was observed in quail of group CX throughout the experiment. Mortality was observed in all other treatment groups (FX, MX, and FM; Figure 1). In group FX, mortality started as early as 3 DPF. Of the 3 birds that died during the first week of the experiment, 2 birds died on d 3 and 1 died on d 4 post-FCM feeding. A total mortality of 2.8% was recorded in the first week, followed by 0.9% in the second week. The mortality in this group was highest (6.6%) during the third week, followed by 1.9% in the last week of the experiment. The overall mortality throughout the course of the experiment in group FX was 12.3%. In group MX, mortality started as early as 2 DPF. A mortality of 4.7% was recorded during the first week, followed by 2.8% in the second week. No mortality was recorded in this group after 11 d of M feeding. The overall mortality throughout the course of the experiment in group MX was 7.6%. In group FM, the highest mortality (12.4%) was recorded during the first week of the experiment and began as early as 2 DPF. Out of a total of 13 birds that died during the first week, 1 bird each died on d 2 and 3, 7 birds died on d 4, and 4 birds died on d 5 post-FCM and post-MCM feeding. A mortality figure of 1.9% was observed in group FM during the second week, followed by 3.8% in the third week and 2.9% in the last week of the experiment. The overall mortality was 20.9% (22 birds), which was higher than the groups fed either FB₁ or M alone.

View larger version (20K): [in this window] [in a new window]   Figure 1. Mortality pattern in Japanese quail fed fumonisin B1 (FX) and moniliformin (MX), either alone or in combination (FM). No mortality was observed in the control group (CX) throughout.

The mean BW of birds at different intervals among groups CX, FX, MX, and FM are shown in Table 2. A steady increase in BW of quail was recorded in all 4 groups throughout the period of the experiment. However, the increase in BW was found to be significantly lower in groups FX, MX, and FM in comparison with group CX. By 28 DPF, BW was only 74.3, 65.1, and 60.9% that of group CX in groups FX, MX, and FM, respectively. Although the mean BW in group FM was found to be lower as compared with groups FX and MX from 14 DPF onward, the difference was significant (P 0.05) only at 28 DPF when compared with group FX only. A significant difference in the mean BW was observed between groups FX and MX at 7 DPF only; thereafter, the values were more or less comparable. A mean treatment effect at the end of the experiment revealed a significant decrease in BW of quail fed FB₁ and M, either alone or in combination, when compared with group CX. The interaction between the different treatments and the age of the quail was also highly significant (P 0.01).

The mean TSP, albumin, and cholesterol concentrations in the sera of Japanese quail from different groups are given in Table 3. In general, the TSP values increased with advancing age of the birds in almost all groups. Although mean serum protein values were higher in groups FX, MX, and FM as compared with group CX at almost all intervals, the difference was only statistically significant (P 0.05) in the combination group FM at 7 and 14 DPF, and in groups MX and FX at 7 and 21 DPF, respectively.

The serum cholesterol values throughout the study were generally higher in the treatment groups (FX, MX, and FM) when compared with those of group CX. The data clearly showed that mean cholesterol values were significantly higher (P 0.05) in groups FX and FM throughout the period of study as compared with those in groups CX and MX. Although serum cholesterol levels were higher in group MX as compared with group CX from 14 DPF throughout the experiment, differences were not statistically significant. The mean age effect was a significant increase in serum cholesterol values up to 21 DPF, followed by a decrease at 28 DPF. The interaction between age and treatment was significant (P 0.01) for both albumin and cholesterol.

Mean values of ALT, AST, and creatinine for each of the experimental groups are presented in Table 4. In general, mean ALT activity in groups FX and FM was higher than that in groups CX and MX throughout the course of the experiment, but the difference in values was significantly higher (P 0.05) only in group FM at 7 DPF and in groups FX and FM at 14 and 21 DPF. The addition of M to the diet (group MX) did not have a significant effect on ALT activity, and the values were comparable to those of group CX throughout the experiment. The mean age effect revealed that ALT activity was maximal at 14 DPF and minimal at 28 DPF.

Mean creatinine values were more or less comparable for groups CX and FX throughout the period of the study. Similarly, mean creatinine values for group FM were not significantly different from those of groups CX and FX up to 21 d of age. At 28 DPF, however, the serum creatinine levels in groups MX and FM were found to be significantly higher than those in groups CX and FX.

Mean values of LDH and CK for each of the experimental groups observed at d 28 postfeeding are given in Table 5. Mean LDH values were significantly higher (P 0.05) in all 3 mycotoxin groups (FX, MX, and FM) as compared with group CX. The mean value in group FM was comparable to those in groups FX and MX. Mean CK values were significantly higher (P 0.05) in groups FX and MX as compared with groups CX and FM.

Skin Thickness. The mean percentage increase in skin thickness recorded in quail from each of the experimental groups at 1, 2, 3, 6, 12, 24, 48, and 72 h after challenge with DNCB is presented in Table 6. In group CX, there was a marked increase in skin thickness up to 3 h, followed by a plateau phase up to 12 h. The maximum thickness was recorded at 24 h, followed by a reduction in thickness at 48 and 72 h postchallenge. In group FX, skin thickness continued to increase gradually up to the end of the observation period, that is, 72 h postchallenge. In group MX, a gradual increase in skin thickness was observed up to 6 h, and it persisted with the same intensity up to 12 h. Another increase in skin thickness was observed in this group at 24 h, which persisted with the same intensity until the end of the experiment. In group FM, skin thickness increased gradually up to 24 h postchallenge. It then decreased slightly at 48 h but showed a peak response at 72 h postchallenge. When mean values were compared among treatment groups, the percentage increase in skin thickness in group FX was significantly lower (P 0.05) compared with group CX at 1, 2, 3, and 24 h, and was lower compared with group MX throughout the study except at 3 and 12 h. The values of group FM fell between those of groups FX and MX but appeared significantly higher (P 0.05) than those of groups CX and FX only at 72 h postchallenge. The mean treatment effect at the conclusion of the experiment across the time period revealed a significantly lower thickness response in group FX as compared with the other groups (CX, MX, and FM). Likewise, the thickness response in group MX as compared with the other groups (CX, FX, and FM) was significantly higher. The mean age effect among different treatments revealed a significant increase in percentage thickness up to 24 h postchallenge.

After Challenge. Up to 6 h, in groups CX and MX the increase in skin thickness (evident as early as 1 h) was due to the accumulation of large amounts of serous exudate and heterophils just below the epithelium. Blood vessels appeared prominent and congested, and hemorrhages were noticed in some places. Lymphocytes started appearing as early as 2 h after challenge and areas close to blood vessels and below the epithelium in both of these groups showed heavy infiltration with lymphocytes. At 3 h postchallenge, there was massive serofibrinous exudate and infiltration mainly with heterophils, but the number of lymphocytes increased significantly in subsequent stages in both of these groups. In contrast, groups FX and FM revealed only slight exudate and a mild inflammatory response with heterophils as the main inflammatory cells up to 6 h, although mild aggregations of lymphocytes could be seen close to blood vessels as early as 2 h post-challenge.

From 12 to 24 h, in comparison with the previous stage, the thickness of the skin in group CX was mainly attributed to an increase in the number of lymphocytes and macrophages. Lymphoid nodules were present in massive numbers throughout. However, a mild serofibrinous exudate was also observed along with occasional infiltration of heterophils. On the other hand, serofibrinous exudate in-filtrated with a massive number of heterophils contributed greatly toward the increase in skin thickness in group FX. The mononuclear inflammatory cell response was poor as compared with group CX at this stage. In group MX, skin response was found to be more cellular and less exudative in all birds at this stage. The mononuclear cell response in group MX was comparatively higher than in group CX at 12 h, whereas it appeared to be more or less comparable to group CX at 24 h. A mild serofibrinous exudate was evident along with scattered heterophils immediately below the epithelium only. In contrast to groups CX, FX, and MX, the increase in skin thickness in group FM was found to be mainly due to excessive accumulation of serofibrinous exudate. A large number of heterophils were observed, whereas the inflammatory response with lymphocytes was more or less comparable to the previous stage and was mild. Few macrophages were evident in the inflammatory reaction in groups FX and FM.

From 48 to 72 h, the histological changes in group CX revealed a purely cellular response, with mononuclear cells being the predominant cells. The exudate was either absent or scanty. A mild serofibrinous exudate accompanied by a massive number of mononuclear cells was observed in group FX at these stages, but leukocyte infiltration was less pronounced as compared with group CX. The histological changes in group MX were more or less similar to those in group CX; however, a mild serofibrinous exudate was still evident. In group FM, the mononuclear cell infiltration was at its peak at 72 h postchallenge as compared with all other previous stages, and heterophils were rarely visible.

Bacteriological Isolations

The liver samples from either dead or killed birds subjected to bacteriological isolations did not reveal any bacteria throughout the course of this study.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Results of the present study showed that feeding of diets amended with 200 ppm of FB₁ and 100 ppm of M led to anorexia, diarrhea, and marked depression in BW. Dark-colored excreta of semisolid consistency occasionally showing mucus was detected as early as 3 d post-FCM feeding and was followed by a large number of birds showing mucus-rich diarrhea up to 2 wk. In previous studies, turkey poults (Weibking et al., 1993b; Ledoux et al., 1996) fed FCM that supplied 25 to 475 mg of FB₁/kg of diet had severe diarrhea after 8 to 10 d; however, no diarrhea was detected in broiler chicks fed FCM supplying up to 525 mg of FB₁/kg from hatch to d 21 (Weibking et al., 1993a). By contrast, diarrhea occurred in quail when 2.5% FCM was incorporated in the chick mash (Deshmukh et al., 2005a), and severe diarrhea with increased mucus content in droppings was observed as early as 5 d in quail fed FCM supplying 300 ppm of FB₁ (Asrani et al., 2006). The histological changes (data not presented) in the intestines from the FCM-fed group in the present study consistently revealed mild to severe goblet cell hyperplasia. The severity of diarrhea in the birds fed FCM reduced significantly after the second week, which can be attributed to the development of tolerance to the mycotoxin with the passage of time (Javed et al., 1993). Diarrhea in the MCM-fed group was discernible as early as 2 DPF and had more of a watery consistency with occasional mucus and subsequently progressing toward the termination of the experiment to fluffy, cotton-like, white, pasty droppings, which appeared to be predominantly urates. Birds in the combination group revealed changes more or less similar to those observed in groups fed FCM or MCM alone. The presence of whitish diarrhea or fluffy, cotton-like, white droppings in the MCM-fed and combination groups, together with an increase in serum creatinine values at 28 d, in the present study suggests that M has some damaging effects on the kidneys. Kidney lesions of varying severity have been reported in M-fed broiler chicks (Ledoux et al., 1995). In an experimental study on 21-d-old broilers fed 546 ppm of FB₁, 98 ppm of FB₂, and 367 ppm of M for 7d, Javed et al. (1993) observed whitish diarrhea that appeared to be predominantly urates and bile in several birds within hours of feeding. Clinical signs of ruffled feathers and stunted growth were observed in almost all the birds fed with culture material-amended diets, either alone or in combination, from 1 wk onward and were consistent with previous studies (Javed et al., 1993; Ledoux et al., 1996; Bermudez et al., 1997; Li et al., 2000b; Asrani et al., 2006). In contrast, Weibking et al. (1993a, b) and Bermudez et al. (1997) showed that feed intake and BW gains of chicks and poults were not affected by dietary FB₁ at levels less than 325 mg of FB₁/kg of feed. However, in the study by Bermudez et al. (1997), both feed intake and BW gain were significantly reduced in turkey poults fed rations containing 100 mg of M/kg. Reductions in feed intake and BW gain were also reported in previous studies with chicks, poults, and ducklings fed 100 mg of M/kg of diet (Javed et al., 1993; Ledoux et al., 1993, 1995; Morris et al., 1997). Reams et al. (1997) reported significant reductions in the BW of birds receiving 80 to 330 mg/kg of M by 8 d. In the present study, feed refusal was comparatively more severe in groups fed either MCM alone or in combination with FCM as compared with the group fed FCM alone, which implies that MCM feeding is specifically responsible for feed refusal, because FB₁-fed birds had normal feed intake. Although BW in the MCM-fed and combination groups were lower than in the group fed FCM alone, no additive or interactive effects were evident between FB₁ and M. In an experimental study using purified FB₁ and M, Javed et al. (1993) also observed that M-fed groups had lower weight gains than FB₁-fed groups. Decreased BW in MCM and the combination group in the present study could be the result of greater feed refusal because of the presence of M in the diet (Li et al., 2000a). Reductions in BW in the FCM-fed group may be linked to a decreased efficiency of feed utilization (Kubena et al., 1997), which was most likely associated with a disruption in sphingolipid biosynthesis by FB₁ (Wang et al., 1991). These sphingolipids are involved in the regulation of cell surface receptors, ion pumps, and other systems vital for cell function and survival (Merrill et al., 1996). The presence of diarrhea and intestinal lesions (goblet cell hyperplasia) may have been additional factors contributing to the reduction in BW in all the culture material-amended groups.

Mortality was found to be highest in quail fed FCM alone during the third week. None of the earlier studies has reported on mortality in either broiler chicks or turkey poults caused by feeding FCM supplying varying levels of FB₁ (Brown et al., 1992; Weibking et al., 1993b; Ledoux et al., 1996). Mortality of 12.3% was recorded in the present study in the group fed FCM alone, which suggests a higher susceptibility of quail to the toxic effects of FB₁ when compared with broiler chicks and turkeys. Previous studies have also shown mortality in quail depending on the level of FCM incorporated into the diet. A mortality of 2.25% was observed in quail when FCM was incorporated at a level of 2.5% (supplying 150 ppm of FB₁) in the ration from d 5 of age (Deshmukh et al., 2005a), whereas mortality was 59% when FCM was added at a level of 5% (supplying 300 ppm of FB₁) in the diet from d 1 (Asrani et al., 2006). Mortality in the group fed MCM alone was observed only up to 11 DPF, and it was highest during the first week, which is consistent with an earlier report by Engelhardt et al. (1989). An overall mortality of 7.6% was observed in the group fed MCM alone. Ledoux et al. (1995) observed 13 and 83% mortality in broiler chicks fed MCM supplying 150 and 300 mg of M/kg, respectively. Javed et al. (1993) reported 40 and 70% mortality, respectively, in chicks fed 27 and 154 mg of pure M/kg. The lower mortality figure can be ascribed to the fact that the source of M in the present study was F. fujikuroi culture material used at a level of 1%, which supplied 100 mg of M/kg of diet. The higher mortality observed by Javed et al. (1993) might be linked to the use of pure M in their studies. It is possible that pure M has a greater bioavailability than the M present in culture material. In culture material, a certain amount of M will be physically trapped in the undigested culture material, and this M has no opportunity to be absorbed by the chick. The overall mortality (20.95%) was found to be highest in the combination group (FCM + MCM) as compared with the groups fed either FCM or MCM alone. Like MCM alone, the mortality in the combination group was also highest (12.4%) during the first week, in contrast to the mortality caused by FB₁ in the present study, which was highest in the third week. Results of a previous study indicating that M may be more toxic than FB₁ to young broilers (Javed et al., 1993) suggest that high mortality in the combination group in the first week might possibly be due to the presence of M in the diet. A greater reduction in feed intake, as observed in the present study in the combination group and the group fed MCM alone, may also partially explain the lower mortality in these groups in the later stages because chicks with reduced feed intake would have ingested less M (Ledoux et al., 1995). A reduction in mortality after 7 DPF in the MCM-fed group may also be linked to the development of cell-mediated immune response because M feeding in the present study did not seem to suppress the immune response and the percentage skin thickness values were also more or less comparable to those of the control group (CX) throughout the duration of experiment. A detailed investigation on this aspect merits serious consideration. In the present study, the interaction between FB₁ and M indicated additive effects in terms of mortality. The histopathological examination of mortality during the first week from these groups (results not presented) clearly suggested that the presence of M in the diet led to severe heart lesions in the combination group, but the presence of FB₁ did not appear to lead to severe liver lesions in the combination group. In previous studies, FB₁ has been shown to be associated with cardiac lesions of mild intensity in turkey poults (Ledoux et al., 1996). The muscle-specific serum chemistry in the present study also suggests that higher levels of CK in the group fed FCM alone may indicate that FB₁ does alter heart function, whereas unaltered levels of serum ALT throughout the period of the experiment in the group fed MCM alone may suggest that M is probably not hepatotoxic. The results of the present study thus suggest that in young quail, the cardiotoxic effect of M may be enhanced in the presence of FB₁ and might eventually lead to high mortality. Similarly, Javed et al. (1993) indicated additive effects in terms of mortality and the onset of clinical signs in chickens fed FB₁ and M. In their studies, a mortality of 37% was observed in chicks fed a combination of 61 mg of FB₁/kg and 66 mg of M/kg. The higher mortality in broiler chicks in their studies could have been because these authors used a higher amount of culture material (5.1 to 45.5%) in their diets compared with the amounts (1.0 to 4.25% culture material) used in the present study, or may indicate a difference in mycotoxin toxicity between quail and chickens.

Biochemical parameters revealed a significant variation between quail fed chick mash alone and those given culture material-amended diets. Total serum protein values were higher in the groups fed MCM, either alone or in combination with FCM, compared with the group fed FCM alone up to 14 DPF, and this phase coincided with the period during which diarrhea was more severe in all treatment groups. During this period, maximum mortality was observed in the combination group and the group fed MCM alone, and the birds in both groups suffered an acute phase of the disease, with heavy mortality within this period. Hence, an increase in TSP with comparatively higher values in the combination group may be attributed to dehydration or to the synthesis of acute phase proteins that occurs 2 to 5 d after tissue injury, inflammation, or necrosis (Duncan et al., 1994). An increase in TSP could also have been caused by an increase in the albumin fraction, the values for which were higher in the combination group as compared with the other groups up to 14 DPF, and this might have occurred concurrently with an increase in globulin concentration during dehydration (Duncan et al., 1994). Both TSP and albumin values were higher in the group fed FCM, either alone or in combination with MCM, compared with the group fed MCM alone after 14 DPF, and this phase coincided with the period during which mortality was higher in the group fed FCM alone; the inflammatory and necrotic lesions in the liver were also severe in this group from 14 DPF onward. Total serum protein and albumin values in the combination group from 14 DPF onward were, however, between those of the groups fed either FCM alone or MCM alone, which could be attributed to reduced feed intake and could have been caused by the mycotoxins. An increase in TSP as a result of feeding FB₁- or M-amended diets has also been reported previously (Bermudez et al., 1996, 1997; Asrani et al., 2006).

Serum ALT and cholesterol levels were found to be significantly higher in the groups fed FCM alone and in the combination group as compared with the other 2 groups. Decreases in serum cholesterol have been reported earlier in FB₁-fed chickens (Weibking et al., 1993a) and turkeys (Weibking et al., 1993b), whereas the values remained unchanged in ducklings (Bermudez et al., 1995). In quail, however, serum cholesterol levels have been shown to be significantly higher following FB₁ feeding, and levels remained elevated throughout the experiment period (Asrani et al., 2006). Increases in the levels of serum cholesterol and ALT in the group fed FCM alone or the combination group at all intervals in the present study could be due to hepatocellular disease (Benjamin, 1985), a result which was further supported by consistent necropsy findings of pale discoloration of the liver observed in both of these groups. In addition, quail have been shown to be highly susceptible to the toxic effects of FB₁, which is characterized by severe hepatic lesions at 150 mg of FB₁/kg of diet (Deshmukh et al., 2005b). The results of the present study clearly suggest that increases in the levels of serum cholesterol and ALT in the combination group were possibly the result of the presence of FCM in the diet because these biochemical parameters remained unchanged in the group fed MCM alone. In a previous study, serum cholesterol levels also remained unchanged with feeding of 100 mg of M/kg to turkeys (Bermudez et al., 1997).

Serum AST, LDH, and CK activity were found to be higher in both the groups fed FCM or MCM alone and in the combination group compared with the group fed chick mash only (CX). Increases in serum AST activity have been observed previously in FB₁-fed broiler chicks (Ledoux et al., 1992), turkeys (Weibking et al., 1993b; Bermudez et al., 1997), and quail (Asrani et al., 2006), and in M-fed chicks (Javed et al., 1995) and turkeys (Bermudez et al., 1997). Significant increases in LDH have also been reported earlier in FB₁- and M-fed chickens (Javed et al., 1995) and turkeys (Bermudez et al., 1997). Significant increases in CK activity in the groups fed FCM and MCM alone suggest that both FB₁ and M are toxic to striated or cardiac muscle. The higher values of LDH and AST in the group fed FCM alone compared with the group fed MCM alone probably reflect significant liver lesions in the presence of FCM because the ALT values remained unchanged in the group fed MCM alone. The only noticeable observation from the AST results was an indication of an interaction between FB₁ and M at 14 DPF, which appeared to be less than additive in nature because the AST values were significantly higher in the combination group than in either the group fed FCM or the group fed MCM alone. After 14 DPF, the values of these biochemical parameters (AST, LDH, and CK) in the combination group were either lower or in between those of groups fed either FCM or MCM alone. The lower values in the combination group compared with the group fed FCM alone could possibly be linked to the greater reduction in feed intake caused by the incorporation of MCM because the quail with reduced feed intake from the third week onward would have ingested less FB₁ and M.

Serum creatinine values were significantly higher in the combination group and in the group fed MCM alone only at 28 DPF, which can be attributed to kidney damage, possibly caused by the presence of M in the diet, because no change in creatinine values was observed in the group fed FCM alone as compared with quail in the CX group. The whitish-colored excreta, which appeared to have an increased urate content in the latter stages of the experiment, suggested involvement of the kidneys in the group fed MCM alone and in the combination group. Kidney lesions of varying severity have been reported earlier in M-fed broiler chicks (Ledoux et al., 1995) and in chickens fed a combination of FB₁ and M (Javed et al., 2005).

In the present study, a delayed type of hypersensitivity reaction was used to study the acquired cellular immune response in vivo. Skin hypersensitivity is an immunological reaction in which antigens cause a release of lymphokines that mediate an inflammatory response, and the extent of inflammation gives the status of cell-mediated immune response. In the current study, groups fed chick mash alone and MCM alone revealed maximum skin thickness at 24 h postchallenge, and the inflammatory response was more or less comparable. Histological studies of these 2 groups revealed massive inflammatory edema in the early stages, usually accompanied by heterophils, but within 24 h, there was the presence of a large number of lymphocytes and macrophages. There was no indication of suppressed cellular immune response by feeding M to quail in the present study; rather, the mononuclear inflammatory response at 12 h postchallenge for the M group was greater compared with all other treatment groups. Results of previous studies, however, have shown a reduction in lymphoid organ weights (bursa, spleen, and thymus) when diets containing varying levels of M were fed to chicks and turkeys (Ledoux et al., 1995; Qureshi et al., 1995; Bermudez et al., 1997; Li et al., 2000a). Decreased bursal weights have been correlated with a reduction in humoral immune response, and broiler chicks and turkey poults fed 100 mg of M/kg of diet revealed significantly lower secondary anti-Newcastle disease virus antibody titers compared with control birds (Li et al., 2000a, b). Significant reductions in white blood cell counts and numbers of viable lymphocytes in earlier studies with chicks fed diets containing M suggested a suppression of cell-mediated immune response in the presence of M (Dombrink-Kurtzman et al., 1993; Javed et al., 1995). However, no immunosuppressive effect of M was evident in the present study. Similarly, the results of a feeding trial conducted by Li et al. (2000a) in broiler chicks showed that lymphocyte proliferation was not affected by 100 mg of M/kg of diet, and the histological changes in spleen, thymus, and bursa in the present studies in the group fed MCM alone did not provide any evidence of lymphoid cell depletions in these organs (data not presented). In comparison with groups fed chick mash alone and MCM alone, both the FCM-fed group and the combination group revealed a gradual increase in skin thickness up to 72 h, and histological studies suggested that the mononuclear inflammatory response was poor until 72 h postchallenge, which could possibly explain the delayed cellular response in these groups. The values for percentage skin thickness and histological response in the combination group were found to fall between those of MCM alone (higher) and FCM alone (lower), which suggested that the poor cell-mediated immune response was possibly due to the presence of FCM (FB₁) in the diet. Earlier studies have shown significantly lower lymphocyte proliferation responses to mitogens in calves (Osweiler et al., 1993) and chicks (Li et al., 1999) fed FB₁. In a previous study, concanavalin A-stimulated proliferation in poults fed 200 mg of FB₁/kg was reduced by 22%, which suggested that T-cell proliferation in poults fed FB₁ was affected (Li et al., 2000b). The low blood lymphocyte counts reported in our recent study with quail chicks also support this finding (Deshmukh et al., 2005a). The mechanism of action of FB₁ has been demonstrated to involve the disruption of complex sphingolipids and accumulation of free sphinganine and sphingosine (Wang et al., 1991). Inhibition of sphingolipid synthesis by FB₁ has been shown to correlate with suppressed cell proliferation (Wang et al., 1991; Yoo et al., 1992). Sphingolipid breakdown products were also recognized as antiproliferative lipids and were demonstrated to decrease cell-mediated and humoral immune response (Felding-Habermann et al., 1990; Hannun and Linardic, 1993; Martinova, 1996). Thus, the poor lymphocyte and macrophage infiltration response in the present study in both groups receiving FCM in the diet might be attributed to depletion of complex sphingolipids and accumulation of free sphinganine and sphingosine caused by FB₁.

Studies on combinations of mycotoxins have received significant attention recently because toxicity of combinations of mycotoxins cannot always be predicted based on their individual toxicities. Further, there is the possibility that one mycotoxin that is relatively nontoxic to poultry might be causing severe damage when present in combination with other mycotoxin(s). Similarly, the presence of one mycotoxin may reverse the damaging effects of other mycotoxins. Previous studies have shown additive toxicity effects with FB₁ and T-2 toxin (Kubena et al., 1995), M and aflatoxin (Kubena et al., 1997), and M and deoxynivalenol (Harvey et al., 1997). With the exception of mortality and serum AST values (at 14 DPF only), no additive or synergistic effects were observed in any response variable parameter measured in the current study, in which all statistical differences were attributed to either one mycotoxin or the other. Minor differences between the results of the present study and previous studies by Javed et al. (1993) and Bermudez et al. (1997) could be due to differences in the susceptibility of quail as compared with broiler chicks and turkeys. Although the results of the present study suggest that FB₁ was the sole cause of immunosuppression and that M appeared to play no role in immunosuppression, additional studies are needed to confirm the phagocyte functional activities in the presence of M. In the present study, however, the presence of M in the diet appeared to reverse the effects of FB₁, particularly at later stages, which may be attributed to significant feed refusal, but further studies are indicated.

	ACKNOWLEDGMENTS

 
The authors are grateful to A. C. Varshney, Dean, College of Veterinary & Animal Sciences, CSK Himachal Pradesh Agricultural University, Palampur, for providing the necessary facilities to undertake this work, and A. J. Bermudez, Fusarium/Poultry Research Laboratory, University of Missouri, Columbia, for his critical review of the manuscript.

Received for publication May 24, 2007. Accepted for publication February 7, 2008.

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