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Mouth Lesions in Broiler Chickens Caused by Scirpenol Mycotoxins1
Mouth Lesions in Broiler Chickens Caused by Scirpenol Mycotoxins ADEDAMOLA A. ADEMOYERO Department of Animal Science, North Carolina A&T State University, Greensboro, North Carolina 27411 PAT B. HAMILTON2 Department of Poultry Science, North Carolina State University, Raleigh, North Carolina 27695-7608 (Received for publication January 14, 1991)
1991 Poultry Science 70:2082-2089 INTRODUCTION
Mouth lesions and associated productivity losses have been a major problem for the poultry industry for the past decade. The best documented cause of mouth lesions in chickens is T-2 toxin [4p\ 15-diacetoxy-8a-(3methyl-butyryloxy)-3a-hydroxy-12,13-epoxytrichothec-9-ene], a trichothecene mycotoxin produced by several Fusarium species (Wyatt et al., 1972a,b). The lesions, which are raised, yellowish-white in color, and sometimes caseous in nature, range from small, round, and pinpoint-sized to large sheets covering most of the mouth. Mouth lesions are about a fourfold more sensitive indicator of T-2 toxicosis than is growth inhibition. The trichothecene mycotoxin, diacetoxyscirpenol [4P,15-diacetoxy-3a-hydroxy-12,13-epoxytrichothec-9-
The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of Ihe products named, nor criticism of similar ones not mentioned. ^To whom correspondence should be addressed.
ene] (DAS), has been reported to cause mouth lesions similar to those caused by T-2 toxin (Chi and Mirocha, 1978; Hoerr et al, 1982a). Another trichothecene mycotoxin that might be involved in mouth lesions is monoacetoxyscirpenol [15-acetoxy-3a,4P-dihydroxy-12,13-epoxy-trichothec-9-ene] (MAS), which was found at 50 ppm along with 2,500 ppm zearalenone in diets containing molded corn and causing mouth lesions (Speers et ah, 1977). Both DAS and MAS are members of the scirpenol family of trichothecene mycotoxins that are acetylated derivatives of the parent alcohol, scirpentriol [3a,4p,15-trihydroxy12,13-epoxytrichothec-9- ene] (STO). The entire family, which includes eight members, has appreciable toxicity at least as great as the T-2 toxin family (Richardson and Hamilton, 1990). Of the eight members, STO, MAS, DAS, and triacetoxyscirpenol [3a,4p,15-triacetoxy12,13-epoxytrichothec-9-ene] (TAS), which differ in degree of acetylation, are available currently in the gram quantities required to evaluate their toxicity in chickens (Richardson
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ABSTRACT Dietary scirpentriol (STO), triacetoxyscirpenol (TAS), monoacetoxyscirpenol (MAS), and diacetoxyscirpenol (DAS), mycotoxins produced by Fusarium species, were compared for their ability to cause mouth lesions when graded dietary levels (0,1,2,4, and 8 \l$ STO or TAS/g; 0,.5,1,2, and 4 |ig MAS or DAS/g) were fed to male broiler chickens for 21 days after hatching. The mourn lesions provoked by each scirpenol were dose-related. The minimum effective doses (MED) were 4 , 2 , 1 , and .5 Hg/g for TAS, STO, DAS, and MAS, respectively, whether the number of affected birds or the number of affected mouth parts (angles, upper beak, lower beak, and tongue) was the measured response. Lesion sites in the mouth varied with the toxin. The rank orders from greatest to least affected sites were angles, upper beak, lower beak, and tongue for TAS and STO, upper beak, lower beak, angles, and tongue for MAS, and upper beak, lower beak, tongue, and angles for DAS. Mouth lesions were clearly visible with each toxin after feeding for 1 wk and the numbers of affected mouth parts almost tripled after 2 wk exposure. During Week 3 of exposure, only the increase caused by MAS was significant (P<.05). The MED for growth inhibition were 2 , 2 , 2 , and 8 ng/g for STO, MAS, DAS, and TAS, respectively. Thus, mouth lesions were of equal or greater sensitivity than growth inhibition as an indicator of scirpenol toxicity. It would appear that the discovery of mouth lesions in birds justifies a mold and mycotoxin control program. (Key words: scirpenols, mouth lesions, Fusarium, dose-response, temporal relationships)
MOUTH LESIONS AND MYCOTOXTNS MATERIALS AND METHODS
Husbandry Day-old, male broiler chicks (Arbor Acres X Arbor Acres) from the university hatchery were housed in electrically heated comfort-zone batteries under continuous lighting. Feed and water were available for ad libitum consumption. The birds were weighed, banded, and assigned randomly to pens in groups of 10 birds per pen. The birds were weighed and examined for mouth lesions weekly for 3 wk. Toxins The mycotoxins were prepared in the authors' laboratory. Crystalline TAS, DAS, and STO were prepared according to Richardson and Hamilton (1987a,b). The MAS was prepared from DAS by the method of Sigg et al. (1965). Purity and identity were established by thin layer and gas chromatography and by comparison with authentic compounds whose identity had been confirmed by mass spectrometry and nuclear magnetic resonance. Feed Feed was a starter mash made by the North Carolina State University feed mill. It contained yellow corn (62.04%), 48.5% protein soybean meal (32.00%), cottonseed oil (2%), salt (.40%), defluorinated phosphate-18% (2.50%), limestone (.60%), DL-methionine (.26%), trace mineral mix (.10%), and vitamin mix (.10%). The absence of STO, MAS, DAS, TAS, and T-2 toxin from the basal diet was established by the chromatographic methods of Richardson et al. (1989). Pure crystalline toxins were added to the diet by dissolving each toxin in acetone (MAS, DAS, and TAS) or in water and acetone (1:9, vol/vol; STO). The solution was added to about 10% of the total feed, which was dried to evaporate the acetone before mixing with the remainder of the feed. Experimental Design A completely randomized experimental design was used to determine the effects of dose and of length of exposure to the toxins on the incidence of mouth lesions. There were four pens of 10 birds per treatment level of each toxin. The dietary concentrations of STO and TAS were 0, 1.0, 2.0, 4.0, and 8.0 ug/g of diet
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and Hamilton, 1987a,b; Ademoyero and Hamilton, 1989). Four different methods have been used to evaluate quantitatively the severity of mouth lesions in chickens. Perhaps the best method is to determine the dry weight of lesions dissected from the mouth (Wyatt et al, 1972b). The dose-response relationship of T-2 toxin to mouth lesions by this method is linear, however, the method is laborious and time consuming. Another method simply notes the number of mouth parts that develop mouth lesions (Speers et al, 1977; Chi and Mirocha, 1978), but the dose-response relationship using this method appears nonlinear (Chi et al, 1977). A method based on a visual scoring system (Huff et al., 1988) has proven useful in studying factors that increase (Kubena et al., 1989a,b) and decrease (Kubena et al, 1990) the toxicity of T-2 toxin. This latter method includes number, site, and size of lesions as criteria in the subjective four-point scoring system. Another approach based on a visual impression without any attempt at measuring or scoring led to the conclusion that severity of mouth lesions was not related to toxin concentration (Hoerr et al^ 1982a). The temporal relationships of mouth lesions evoked by trichothecene mycotoxins are important in the industrial setting, but the subject has been investigated only slightly. Wyatt et al. (1972b) reported that mouth lesions first appeared after exposure to T-2 toxin for about 1 wk, but Hoerr et al. (1982a) stated the onset of lesions ranged from 2 to 4 days postexposure. Chi and Mirocha (1978) observed lesions on Day 5 of exposure. Chi et al. (1977) did not see lesions in broilers fed T-2 toxin from hatching until after 2 wk of exposure. Hoerr et al. (1982a) stated the size and number of lesions increased through 14 days of exposure but decreased thereafter. Chi et al. (1977) reported that the percentage of birds having lesions in any part of the mouth increased through 21 days and remained essentially constant through 63 days of exposure. The objectives of the present study were 1) to compare the dose-response relationships of STO, MAS, DAS, and TAS, scirpenols differing in degree of acetylation, in causing mouth lesions; 2) to compare the temporal relationships of the mouth lesions elicited; and 3) to gain insight into whether mouth lesions might be considered a general attribute of the trichothecene toxins produced by Fusarium species.
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ADEMOYERO AND HAMILTON TABLE 1. Body weights of chickens fed graded concentrations of scirpenol mycotoximr
Concentration Oxg/g) 0 .5 1.0 2.0 4.0 8.0
TAS
STO
610"
599"
62 l a 599 a 627 a 561 b
593 a 556 b 554 b 450°
MAS
DAS
M \S) a 604 609a 588 a 518 b 441°
603 a 608 a 585 a 549 b 441°
a_c
Values in a column with no common superscripts differ significantly (P<05). Values are the means of four groups of 10 birds fed each indicated level of each toxin for 3 wk. TAS = triacetoxyscirpenol; STO = scirpentriol; MAS = monoacetoxyscirpenol; and DAS = diacetoxyscirpenol.
Statistical Analyses The body weights associated with dose levels of a toxin were subjected to a one-way analysis of variance in which an F ratio was calculated. If it were significant (P<.05), the least significant difference (t test) among the treatment means was calculated (Bruning and Kintz, 1977). For each toxin, the number of lesions on the four parts of the mouth (tongue, upper beak, lower beak, and angles) were subjected to a logit transformation prior to an analysis of variance based on a factorial design with dose (five levels) and time (three levels) as factors. Then F ratios were calculated and if significant (P<.05), the means were separated by Duncan's multiple range test using the General Linear Models procedure of SAS® (SAS Institute, 1982). The numbers of birds and the numbers of mouth parts affected by the different doses of MAS were subjected to linear regression analysis. RESULTS
The minimum effective dose (MED) for growth inhibition by STO, MAS, and DAS was 2 ug/g diet (Table 1). Considerably less
growth inhibition occurred with the fully acetylated TAS, whose MED was 8.0 u.g/g diet. Duration of exposure to the dietary scirpenols had significant (P<.05) effects on the incidence of mouth lesions (Table 2). Lesions were obvious in the chicks after 1 wk of exposure to the four individual toxins (unscheduled examination of the birds on the highest toxin level at 4 days of age did not reveal lesions), and the total number of lesions essentially tripled by the end of 2 wk of exposure to the toxins. Only MAS caused a significant (P<.05) increase in total lesions during the 3rd wk. When the time data were examined for the abilities of the toxins to cause lesions on the different mouth parts, striking differences were observed. For example, most (53%) of the lesions caused by TAS occurred in the angles of the mouth, but no tongue lesions were caused by this toxin. The lesions caused by DAS presented a quite different partem with only 6% occurring on the angles, 47% inside the upper beak, 32% inside the lower beak, and 15% on the tongue. Although tongue lesions were considerably more likely to be caused by DAS than by the other scirpenols, none of the scirpenols had a great effect on the tongue. The partem of lesions caused by STO was very similar to that of TAS with the rank order from most common to least common being angles, upper beak, lower beak, and tongue. The partem for MAS resembled that for DAS with the rank order for lesions being upper beak, lower beak, angles, and tongue. The dose-response relationships of the mouth lesions caused by the scirpenols differed markedly from those found for growth inhibition. When the total number of mouth
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and the concentrations of MAS and DAS were 0, .5,1.0,2.0, and 4.0 ug/g of diet. The four toxins were investigated in a single study conducted in one room at the same time. At the end of Weeks 1, 2, and 3, each bird was examined for the presence of lesions on four mouth parts. The presence or absence of a lesion on the tongue, inside of the upper beak, inside of the lower beak, and on the angles (the two angles together were considered to be one mouth part) of the mouth was recorded on a treatment level basis. No attempt was made to assess the severity of the lesions, just their presence on the indicated mouth parts.
MOUTH LESIONS AND MYCOTOXINS
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TABLE 2. Effect of exposure time on incidence of lesions on mouth parts of chickens fed Lesion site
Time
scirpenolr
TAS
STO
MAS
DAS
6b 16** 26 a (46) 9b
5a 22a 24 a (24) 13b
la 9s 6\6) 19b
19 a 18 a (32)
31ab 41 a (42)
50s 51 a (47)
0" 3a 8 a (14) 0b
3b 14ab 28 a (29) 2a
3b 16* 35 a (32) 3a
0b 4 a (8)
6a 5 a (5)
10s 16 a (15)
15 b 38 a 56"(100)
23c 73b 98 a (100)
26b 85a 108 a (100)
(wk) 1 2 3
4" 19 s 19a(53)2
Upper beak
1 2 3
6" 14a 13a(36)
Lower beak
1 2 3
0" 0* 4 a (ll)
Tongue
1 2 3
0" 0" (PCO)
1 2 3
10b 33" 36 a (100)
Total mouth parts with lesions
a-c Values in a column within a lesion site with no common superscripts differ significantly (P<.05). Values are based on all birds fed the indicated toxin.
'TAS = triacetoxyscirpenol; STO = scirpentriol; MAS = monoacetoxyscirpenol; and D A S = diacetoxyscirpenol. t a b u l a r values in parentheses are the numbers of a particular mouth part having lesions expressed as the percentage of the total number of the mouth part exhibiting lesions after 3 wk exposure to the particular toxin. For example, TAS caused 36 total mouth parts to have lesions after 3 wk and 19 of them were in the angles o f the mouth, giving an incidence of 53% ([19 + 36] x 100) of the lesions occurring in the angles. Numbers are based on all birds receiving the toxin, i.e., the numbers for all treatment levels of the toxin were summed.
parts having lesions were considered, the MED were .5, 1.0, 2.0, and 4.0 u.g/g for MAS, DAS, STO, and TAS, respectively, giving an eightfold difference between MAS, the most effective, and TAS, the least effective (Table 3). When the different parts of the mouth were considered, there were no significant (P<.05) dose effects of TAS and STO on the lower beak or tongue nor of MAS on the angles. When the part of the mouth on which the toxins exhibited the greatest effect was considered, the MED of TAS and STO on the angles were 8.0 and 4.0 |Xg/g, respectively, and the MED of MAS and DAS on the upper beak were 2.0 and 1.0 u.g/g, respectively. When the dose-response relationships of the lesions were considered, with the number of affected birds being the response (Table 4), the pattern was the same as that obtained when total number of mouth parts having lesions was the response. The MED were .5, 1.0, 2.0, and 4.0 u.g/g for MAS, DAS, STO, and TAS, respectively, the same values obtained when the total number of mouth parts having lesions were considered. The fact that each dose of MAS gave a response (total number of affected mouth parts or birds) that differed significantly (P<.05)
from the responses of the other doses (Tables 3 and 4) permitted a meaningful linear regression of the MAS data. When Y = number of affected birds and X = log2 of MAS concentration, the equation was Y = 3.2 + 7. IX (r = .98). When Y = number of affected mouth parts, the equation became Y = -.8 + 19.8X (r = .99). DISCUSSION
The time of exposure required to provoke mouth lesions was clearly 1 wk or less for all four scirpenols (Table 2). The number of mouth parts having lesions about tripled from the end of the 1st wk until the end of the 2nd wk with each toxin, but only MAS caused a significant (P<.05) increase during the 3rd wk. These findings agree well with those of Wyatt et al. (1972b), who observed onset of T-2 induced lesions at about 1 wk, and with those of Chi and Mirocha (1978), who observed onset at 5 days. In contrast, Hoerr et al. (1982a) found onset to be between 2 and 4 days and Chi et al. (1977) found onset at 2 wk of exposure. Nevertheless, the bulk of the data supports the conclusion that exposure for only a few days results in mouth lesions.
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Angles
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ADEMOYERO AND HAMILTON TABLE 3. Dose-responses of mouth lesions in broilers fed scirpenolr
Lesion site Angles
Upper beak
Dose (Hg/g diet) 0 .5 1 2 4 8 0 .5 1 2 4 8
Lower beak
TAS
MAS
DAS
i'
0° 12 ab 24" lc jbc gab lla 13" 0* 1* 3a 0* 0*
0* 0* 2a 8a 0"
'6*'
'6"'
0* 0* 0s
2a la la
0°
0°
0° llb 28 a
jab 18a 27 a
0° 0°b
10*
5 3bc 21*
16a 32° 58 b 83*
6° 7° 37 b 64'ab 1051
8C
Total
23D
26 a 36a
2Qab 44 a
2bc
a e ~ Values in a column within a lesion site with no common superscripts differ significantly (P<05). Values are based on all birds fed each dose of each toxin for 3 wk. J TAS = triacetoxyscirpenol; STO = scirpentriol; MAS = monoacetoxyscirpenol; and DAS = diacetoxyscirpenol.
The current findings of small, if any, increases in the number of mouth parts with lesions between the 2nd and 3rd wk of exposure agree with the results of Chi et al. (1977), who found a constant percentage of affected birds from 3 to 9 wk exposure. The current findings do not agree with the findings of Hoerr et al. (1982a), who observed that the size and number of oral lesions increased through 14 days exposure and decreased thereafter, nor do the current findings agree with their conclusion that oral mucosa becomes resistant after 14 days of exposure. Also contrary to their conclusions are field observations that mouth lesion problems in broiler breeder flocks continue for months (P. B. Hamilton, unpublished data) and laboratory observations that mouth lesions induced by
DAS in broiler breeders continue until after DAS is removed from the feed (R. Sigmon, J. Brake, and P. B. Hamilton, Dept. of Poultry Science, N. C. State University, Raleigh, NC, unpublished data). The latter two observations, the current findings, and those of Chi et al. (1977), support a conclusion that mouth lesions will not disappear until the responsible mycotoxin or toxins are removed from the feed. The locations of the lesion sites in the mouth have practical implications, because the locations of lesion sites in field outbreaks are not randomly dispersed (P. B. Hamilton, unpublished data). None of the scirpenols caused lesions on only a single part of the mouth, but the lesions caused by TAS and STO were mainly on the angles of the mouth
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6* Tongue
STO
(Number of mouth parts with lesions) 0" 5* 2a c 0b 5a 0b 16a 11* a 5* 16 lla 13a 13a 16a 6° 5C b 23 b 2 15° a s 38 ab 31 10 32 a 48 a 13a 19 s
MOUTH LESIONS AND MYCOTOXtNS
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TABLE 4. Frequency of mouth lesions in chickens fed scirpenol mycotoxinr Dose (ug/g diet) 0 .5 1 2 4 8
TAS l/39 b ... 3/40b 4/40b 12/39a 15/39"
STO
MAS
DAS
(Number of birds with lesions/total number of birds) 4/40° 3/38e 2/40° ... 8/38d 3/40° 3/40° 21/39c 19/40b ll/40 b 25/38 b 24/40 ab 13/39ab 30/36a 32/39" 21/39" ^^ ._^
a_e Values in a column with no common superscripts differ significantly (P<.05). Values are affected birds/birds exposed to indicated toxin for 3 wk. TAS = triacetoxyscirpenol; STO = scirpentriol; MAS = monoacetoxyscirpenol; and DAS = diacetoxysciipenol.
In the current study, the MED and the dose levels of a particular toxin that produced significantly (P<.05) different responses were identical whether the number of affected birds (Table 4) or the number of affected mouth parts (Table 3) was the dependent variable. Because it is easier to count affected birds rather than affected mouth parts, counting affected birds would probably be the preferred way of quantitatively analyzing the ability of trichothecenes to cause mouth lesions. When the toxicities of the scirpenols were compared using the number of affected mouth parts (Table 3) or of affected birds (Table 4) to determine MED, the rank order from greatest to least toxicity was MAS, DAS, STO, and TAS. This was quite different from the rank order MAS, DAS, and STO, which were greater than TAS when body weight was the criterion of toxicity (Table 1). The ratios between the MED for body weight and mouth lesions were 4, 2, 1, and 2 for MAS, DAS, STO, and TAS, respectively, indicating that mouth lesions were of equal or greater sensitivity as an indicator of scirpenol toxicoses than was body weight. The same relationship was observed with T-2 toxin, which belongs to another family of trichothecene toxins (Wyatt et al, 1972a, 1973). The dose-response relationships of the toxins in the present study, except for MAS, cannot be described in detail because of the generally small number of doses having a significant (P<.05) effect on the response. However, MAS, which satisfied the criterion of each dose level producing a response significantly different (P<.05) from the other dose levels, yielded linear regression equations with r>.98. These results imply that the doseresponse relationship of MAS, at least, is
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and rarely on the tongue. The derivatives, MAS and DAS, caused their greatest effect on the upper beak, followed by the lower beak, and least effect on the tongue, although DAS showed a greater effect on the tongue than the other scirpenols. These findings support conclusions that concentration of lesions in different parts of the mouth in different outbreaks is not inconsistent with assigning the cause to toxicosis produced by Fusarium species and that the predominant location of the lesions can be useful in assigning the cause to specific mycotoxins. The present findings on lesion sites are not in total agreement with some earlier observations. Speers et al. (1977) found essentially an equal number of lesions on the angles, mandibles, and tongues of birds fed MAS and zearalenone. Chi and Mirocha (1978) found in birds fed DAS essentially equal percentages of upper beaks (100%) and tongues (90%) bearing lesions but the lower beaks and angles had about half those percentages. Hoerr et al. (1982a) offered no numbers but stated that all mouth parts were affected and that many lesions were near the salivary duct openings of birds fed DAS. A concentration of mouth lesions in and surrounding salivary duct openings has been seen in field outbreaks (P. B. Hamilton, unpublished data), but only a few such lesions were seen in the current study. Hoerr et al. (1982a) offered as one explanation for such lesions that T-2 toxin was excreted in the saliva, presumably after gastrointestinal absorption. Because birds intubated with DAS did not have mouth lesions (Hoerr et al., 1981, 1982b), their other explanation that toxincontaining feed becomes moistened with saliva and adheres to and around the duct seems more likely.
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ADEMOYERO AND HAMILTON
The discrepancies between the present study and the studies cited earlier appear to have several causes. It can be calculated from the data of Speers et al. (1977) that the moldy com they used as the source of MAS contained 5% zearalenone. This seems very unlikely. If a decimal error is assumed, giving a more likely .5% zearalenone, then their reported level of 50 ug MAS/g diet becomes 5 ug/g and their growth and lesion frequency data essentially agree with the present study. It must be mentioned that their corn was molded with Fusarium roseum 'Gibbosum', which thenlaboratory later reported to produce DAS and its two isomers, 3,4-DAS and 3,15-DAS, as well as MAS and zearalenone (Mirocha et al., 1985). A mixture of these scirpenols and zearalenone might well give the peculiar pattern of lesion locations observed by Speers et al. (1977), i.e., approximately equal numbers of lesions on each mouth part. Hoerr et al. (1982a), who found that the concentration of DAS did not influence lesion severity and that the number and size of lesions peaked at 14 days exposure and declined thereafter, added crystalline DAS to a small amount of casein as a carrier and combined it with a commercial broiler starter mash instead of dissolving the crystals in a solvent and adding the solution to the diet. It is difficult to disperse crystalline material evenly at parts per million levels in feed, and even if good dispersion is achieved,
segregation can occur readily. Vitamins and trace minerals usually are added to diets as premixes in order to avoid such problems. Inadequate dispersion and segregation might well account for the failure of Hoerr et al. (1982a) to obtain a dose-related response. In addition to these problems, Speers et al. (1977), Chi and Mirocha (1978), and Hoerr et al. (1982a) used small numbers of birds (5 to 20) per treatment and few treatment levels (one to two) and frequently were unable to analyze their data statistically. The ability of the four scirpenols differing in degree of acetylation to induce mouth lesions in a dose-related manner along with the prior reported ability of T-2 toxin (Wyatt et al, 1972a,b, 1973) imply that trichothecenes in general might cause mouth lesions and associated productivity losses in poultry. Certainly, the current findings support the contention that the finding of mouth lesions in birds is ample reason to commence a mold and mycotoxin control program in poultry operations or to examine an existing control program. ACKNOWLEDGMENTS
The authors thank Hunter Edwards and Nancy Bailey for technical assistance. The research was supported in part by Cambridge Products, Lie, Springfield, MO 68501. REFERENCES Ademoyero, A. A., and P. B. Hamilton, 1989. Influence of degree of acetylation of scirpenol mycotoxins on feed refusal by chickens. Poultry Sci. 68:854-855. Bruning, J. L., and B. L. Kintz, 1977. Computational Handbook of Statistics, 2nd ed. Scott, Foresman and Co. Glenview, IL. Chi, M. S., and C. J. Mirocha, 1978. Necrotic oral lesions in chickens fed diacetoxyscirpenol, T-2 toxin, and crotocin. Poultry Sci. 57:807-808. Chi, M S., C. J. Mirocha, H. J. Kurtz, G. Weaver, F. Bates, and W. Shimoda, 1977. Subacute toxicity of T-2 toxin in broiler chicks. Poultry Sci. 56:306-313. Hoerr, F. J., W. W. Carlton, and B. Yagen, 1981. Mycotoxicosis caused by a single dose of T-2 toxin or diacetoxyscirpenol in broiler chickens. Vet Pathol. 18:652-664. Hoerr, F. J., W. W. Carlton, B. Yagen, and A. Z. Joffe, 1982a. Mycotoxicosis caused by either T-2 toxin or diacetoxyscirpenol in the diet of broiler chickens. Fundam. Appl. Toxicol. 2:121-124. Hoerr, F. J., W. W. Carlton, B. Yagen, and A. Z. Joffe, 1982b. Mycotoxicosis produced in broiler chickens by multiple doses of either T-2 toxin or diacetoxyscirpenol. Avian Pathol. 11:369-383. Huff, W. E., R. B. Harvey, L. F. Kubena, and G. E. Rottinghaus, 1988. Toxic synergism between afla-
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linear, in agreement with data for T-2 toxin (Wyatt et al., 1972b). Previous studies on DAS and MAS in chickens are inadequate for comparing with our dose-response data. Chi and Mirocha (1978) observed 100% of the 10 birds fed 5 ug DAS/g diet to have mouth lesions coupled with growth depression of 25%. The present comparable values by interpolation were about 85 and 28%, respectively. Further comparison cannot be made because Chi and Mirocha (1978) used only the one level of DAS. Hoerr et al. (1982a), who studied two levels of DAS (4 and 16 ug/g), stated without offering data that the concentration of DAS did not influence mouth lesion severity. However, they did state that 16 but not 4 ug DAS/g diet inhibited growth significantly (P<.05), again without offering data. Speers et al. (1977) reported that 25 and 50 ug MAS/g diet inhibited growth 22 and 28%, respectively, and each caused five of six birds, to have mouth lesions. With 4 ug MAS/g, 27% growth inhibition was obtained and 30 of 36 birds had mouth lesions.
MOUTH LESIONS AND MYCOTOXINS
13495. Appl. Enviroa Microbiol. 53:457-^59. Richardson, K. E., and P. B. Hamilton, 1990. Comparative toxicity of scirpentriol and its acetylated derivatives. Poultry Sci. 69:397-402. Richardson, K. E., G. E. Toney, C. A. Haney, and P. B. Hamilton, 1989. Occurrence of scirpentriol and its seven acetylated derivatives in culture extracts of Fusarium sambucinum NRRL 13495. J. Food Prot 52:871-876. SAS Institute, 1982. SAS® User's Guide. 1982 ed. SAS Institute, Inc., Cary, NC. Sigg, H. P., R. Mauli, E. Flury, and D. Hauser, 1965. Die Konstitution von Diacetoxyscirpenol. Helv. Chim. Acta 48:962-988. Speers, G. M, C. J. Mirocha, C. M. Christensen, and J. C. Behrens, 1977. Effects on laying hens of feeding corn invaded by two species of Fusarium and pure T-2 mycotoxin. Poultry Sci. 56:98-102. Wyatt, R. D., J. R. Harris, P. B. Hamilton, and H. R. Burmeister, 1972a. Possible field outbreaks of fusariotoxicosis in avians. Avian Dis. 16:1123-1129. Wyatt, R. D., B. A. Weeks, P. B. Hamilton, and H. R. Burmeister, 1972b. Severe oral lesions in chickens caused by ingestion of dietary fnsariotoxin T-2. Appl. Microbiol. 24:251-257. Wyatt, R. D., P. B. Hamilton, and H. R. Burmeister, 1973. The effects of T-2 toxin in broiler chickens. Poultry Sci. 52:1853-1859.
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toxin and T-2 toxin in broiler chickens. Poultry Sci. 67:1418-1423. Kubena, L. F., R. B. Harvey, W. E. Huff, D. E. Corrier, T. D. Phillips, and G. E. Rottinghaus, 1989a. Influence of ochratoxin (OA) and T-2 toxin singly and in combination on broiler chickens. Poultry Sci. 68: 867-872. Kubena, L. F., W. E. Huff, R. B. Harvey, T. D. Phillips, and G. E. Rottinghaus, 1989b. Individual and combined toxicity of deoxynivalenol and T-2 toxin in broiler chicks. Poultry Sci. 68:622-626. Kubena, L. F., R. B. Harvey, W. E. Huff, D. E. Corrier, T. D. Phillips, and G. E. Rottinghaus, 1990. Efficacy of a hydrated sodium calcium aluminosilicate to reduce the toxicity of aflatoxin and T-2 toxin. Poultry Sci. 69:1078-1086. Mirocha, C. J., R. J. Pawlosky, T. X. Zhu, and Y. W. Lee, 1985. Chemistry and biological activity of Fusarium roseum mycotoxins. Pages 291-307 in: Trichothecenes and Other Mycotoxins. J. Lacey, ed. John Wiley and Sons, New York, NY. Richardson, K. E., and P. B. Hamilton, 1987a. Preparation of 4,15-diacetoxyscirpenol from cultures of Fusarium sambucinum NRRL 13495. Appl. Environ. Microbiol. 53:460-462. Richardson, K. E., and P. B. Hamilton, 1987b. Preparation of scirpentriol and triacetoxyscirpenol in good yield from cultures of Fusarium sambucinum NRRL
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1991 Poultry Science 70:2082-2089 INTRODUCTION
Mouth lesions and associated productivity losses have been a major problem for the poultry industry for the past decade. The best documented cause of mouth lesions in chickens is T-2 toxin [4p\ 15-diacetoxy-8a-(3methyl-butyryloxy)-3a-hydroxy-12,13-epoxytrichothec-9-ene], a trichothecene mycotoxin produced by several Fusarium species (Wyatt et al., 1972a,b). The lesions, which are raised, yellowish-white in color, and sometimes caseous in nature, range from small, round, and pinpoint-sized to large sheets covering most of the mouth. Mouth lesions are about a fourfold more sensitive indicator of T-2 toxicosis than is growth inhibition. The trichothecene mycotoxin, diacetoxyscirpenol [4P,15-diacetoxy-3a-hydroxy-12,13-epoxytrichothec-9-
The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of Ihe products named, nor criticism of similar ones not mentioned. ^To whom correspondence should be addressed.
ene] (DAS), has been reported to cause mouth lesions similar to those caused by T-2 toxin (Chi and Mirocha, 1978; Hoerr et al, 1982a). Another trichothecene mycotoxin that might be involved in mouth lesions is monoacetoxyscirpenol [15-acetoxy-3a,4P-dihydroxy-12,13-epoxy-trichothec-9-ene] (MAS), which was found at 50 ppm along with 2,500 ppm zearalenone in diets containing molded corn and causing mouth lesions (Speers et ah, 1977). Both DAS and MAS are members of the scirpenol family of trichothecene mycotoxins that are acetylated derivatives of the parent alcohol, scirpentriol [3a,4p,15-trihydroxy12,13-epoxytrichothec-9- ene] (STO). The entire family, which includes eight members, has appreciable toxicity at least as great as the T-2 toxin family (Richardson and Hamilton, 1990). Of the eight members, STO, MAS, DAS, and triacetoxyscirpenol [3a,4p,15-triacetoxy12,13-epoxytrichothec-9-ene] (TAS), which differ in degree of acetylation, are available currently in the gram quantities required to evaluate their toxicity in chickens (Richardson
2082
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ABSTRACT Dietary scirpentriol (STO), triacetoxyscirpenol (TAS), monoacetoxyscirpenol (MAS), and diacetoxyscirpenol (DAS), mycotoxins produced by Fusarium species, were compared for their ability to cause mouth lesions when graded dietary levels (0,1,2,4, and 8 \l$ STO or TAS/g; 0,.5,1,2, and 4 |ig MAS or DAS/g) were fed to male broiler chickens for 21 days after hatching. The mourn lesions provoked by each scirpenol were dose-related. The minimum effective doses (MED) were 4 , 2 , 1 , and .5 Hg/g for TAS, STO, DAS, and MAS, respectively, whether the number of affected birds or the number of affected mouth parts (angles, upper beak, lower beak, and tongue) was the measured response. Lesion sites in the mouth varied with the toxin. The rank orders from greatest to least affected sites were angles, upper beak, lower beak, and tongue for TAS and STO, upper beak, lower beak, angles, and tongue for MAS, and upper beak, lower beak, tongue, and angles for DAS. Mouth lesions were clearly visible with each toxin after feeding for 1 wk and the numbers of affected mouth parts almost tripled after 2 wk exposure. During Week 3 of exposure, only the increase caused by MAS was significant (P<.05). The MED for growth inhibition were 2 , 2 , 2 , and 8 ng/g for STO, MAS, DAS, and TAS, respectively. Thus, mouth lesions were of equal or greater sensitivity than growth inhibition as an indicator of scirpenol toxicity. It would appear that the discovery of mouth lesions in birds justifies a mold and mycotoxin control program. (Key words: scirpenols, mouth lesions, Fusarium, dose-response, temporal relationships)
MOUTH LESIONS AND MYCOTOXTNS MATERIALS AND METHODS
Husbandry Day-old, male broiler chicks (Arbor Acres X Arbor Acres) from the university hatchery were housed in electrically heated comfort-zone batteries under continuous lighting. Feed and water were available for ad libitum consumption. The birds were weighed, banded, and assigned randomly to pens in groups of 10 birds per pen. The birds were weighed and examined for mouth lesions weekly for 3 wk. Toxins The mycotoxins were prepared in the authors' laboratory. Crystalline TAS, DAS, and STO were prepared according to Richardson and Hamilton (1987a,b). The MAS was prepared from DAS by the method of Sigg et al. (1965). Purity and identity were established by thin layer and gas chromatography and by comparison with authentic compounds whose identity had been confirmed by mass spectrometry and nuclear magnetic resonance. Feed Feed was a starter mash made by the North Carolina State University feed mill. It contained yellow corn (62.04%), 48.5% protein soybean meal (32.00%), cottonseed oil (2%), salt (.40%), defluorinated phosphate-18% (2.50%), limestone (.60%), DL-methionine (.26%), trace mineral mix (.10%), and vitamin mix (.10%). The absence of STO, MAS, DAS, TAS, and T-2 toxin from the basal diet was established by the chromatographic methods of Richardson et al. (1989). Pure crystalline toxins were added to the diet by dissolving each toxin in acetone (MAS, DAS, and TAS) or in water and acetone (1:9, vol/vol; STO). The solution was added to about 10% of the total feed, which was dried to evaporate the acetone before mixing with the remainder of the feed. Experimental Design A completely randomized experimental design was used to determine the effects of dose and of length of exposure to the toxins on the incidence of mouth lesions. There were four pens of 10 birds per treatment level of each toxin. The dietary concentrations of STO and TAS were 0, 1.0, 2.0, 4.0, and 8.0 ug/g of diet
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and Hamilton, 1987a,b; Ademoyero and Hamilton, 1989). Four different methods have been used to evaluate quantitatively the severity of mouth lesions in chickens. Perhaps the best method is to determine the dry weight of lesions dissected from the mouth (Wyatt et al, 1972b). The dose-response relationship of T-2 toxin to mouth lesions by this method is linear, however, the method is laborious and time consuming. Another method simply notes the number of mouth parts that develop mouth lesions (Speers et al, 1977; Chi and Mirocha, 1978), but the dose-response relationship using this method appears nonlinear (Chi et al, 1977). A method based on a visual scoring system (Huff et al., 1988) has proven useful in studying factors that increase (Kubena et al., 1989a,b) and decrease (Kubena et al, 1990) the toxicity of T-2 toxin. This latter method includes number, site, and size of lesions as criteria in the subjective four-point scoring system. Another approach based on a visual impression without any attempt at measuring or scoring led to the conclusion that severity of mouth lesions was not related to toxin concentration (Hoerr et al^ 1982a). The temporal relationships of mouth lesions evoked by trichothecene mycotoxins are important in the industrial setting, but the subject has been investigated only slightly. Wyatt et al. (1972b) reported that mouth lesions first appeared after exposure to T-2 toxin for about 1 wk, but Hoerr et al. (1982a) stated the onset of lesions ranged from 2 to 4 days postexposure. Chi and Mirocha (1978) observed lesions on Day 5 of exposure. Chi et al. (1977) did not see lesions in broilers fed T-2 toxin from hatching until after 2 wk of exposure. Hoerr et al. (1982a) stated the size and number of lesions increased through 14 days of exposure but decreased thereafter. Chi et al. (1977) reported that the percentage of birds having lesions in any part of the mouth increased through 21 days and remained essentially constant through 63 days of exposure. The objectives of the present study were 1) to compare the dose-response relationships of STO, MAS, DAS, and TAS, scirpenols differing in degree of acetylation, in causing mouth lesions; 2) to compare the temporal relationships of the mouth lesions elicited; and 3) to gain insight into whether mouth lesions might be considered a general attribute of the trichothecene toxins produced by Fusarium species.
2083
2084
ADEMOYERO AND HAMILTON TABLE 1. Body weights of chickens fed graded concentrations of scirpenol mycotoximr
Concentration Oxg/g) 0 .5 1.0 2.0 4.0 8.0
TAS
STO
610"
599"
62 l a 599 a 627 a 561 b
593 a 556 b 554 b 450°
MAS
DAS
M \S) a 604 609a 588 a 518 b 441°
603 a 608 a 585 a 549 b 441°
a_c
Values in a column with no common superscripts differ significantly (P<05). Values are the means of four groups of 10 birds fed each indicated level of each toxin for 3 wk. TAS = triacetoxyscirpenol; STO = scirpentriol; MAS = monoacetoxyscirpenol; and DAS = diacetoxyscirpenol.
Statistical Analyses The body weights associated with dose levels of a toxin were subjected to a one-way analysis of variance in which an F ratio was calculated. If it were significant (P<.05), the least significant difference (t test) among the treatment means was calculated (Bruning and Kintz, 1977). For each toxin, the number of lesions on the four parts of the mouth (tongue, upper beak, lower beak, and angles) were subjected to a logit transformation prior to an analysis of variance based on a factorial design with dose (five levels) and time (three levels) as factors. Then F ratios were calculated and if significant (P<.05), the means were separated by Duncan's multiple range test using the General Linear Models procedure of SAS® (SAS Institute, 1982). The numbers of birds and the numbers of mouth parts affected by the different doses of MAS were subjected to linear regression analysis. RESULTS
The minimum effective dose (MED) for growth inhibition by STO, MAS, and DAS was 2 ug/g diet (Table 1). Considerably less
growth inhibition occurred with the fully acetylated TAS, whose MED was 8.0 u.g/g diet. Duration of exposure to the dietary scirpenols had significant (P<.05) effects on the incidence of mouth lesions (Table 2). Lesions were obvious in the chicks after 1 wk of exposure to the four individual toxins (unscheduled examination of the birds on the highest toxin level at 4 days of age did not reveal lesions), and the total number of lesions essentially tripled by the end of 2 wk of exposure to the toxins. Only MAS caused a significant (P<.05) increase in total lesions during the 3rd wk. When the time data were examined for the abilities of the toxins to cause lesions on the different mouth parts, striking differences were observed. For example, most (53%) of the lesions caused by TAS occurred in the angles of the mouth, but no tongue lesions were caused by this toxin. The lesions caused by DAS presented a quite different partem with only 6% occurring on the angles, 47% inside the upper beak, 32% inside the lower beak, and 15% on the tongue. Although tongue lesions were considerably more likely to be caused by DAS than by the other scirpenols, none of the scirpenols had a great effect on the tongue. The partem of lesions caused by STO was very similar to that of TAS with the rank order from most common to least common being angles, upper beak, lower beak, and tongue. The partem for MAS resembled that for DAS with the rank order for lesions being upper beak, lower beak, angles, and tongue. The dose-response relationships of the mouth lesions caused by the scirpenols differed markedly from those found for growth inhibition. When the total number of mouth
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and the concentrations of MAS and DAS were 0, .5,1.0,2.0, and 4.0 ug/g of diet. The four toxins were investigated in a single study conducted in one room at the same time. At the end of Weeks 1, 2, and 3, each bird was examined for the presence of lesions on four mouth parts. The presence or absence of a lesion on the tongue, inside of the upper beak, inside of the lower beak, and on the angles (the two angles together were considered to be one mouth part) of the mouth was recorded on a treatment level basis. No attempt was made to assess the severity of the lesions, just their presence on the indicated mouth parts.
MOUTH LESIONS AND MYCOTOXINS
2085
TABLE 2. Effect of exposure time on incidence of lesions on mouth parts of chickens fed Lesion site
Time
scirpenolr
TAS
STO
MAS
DAS
6b 16** 26 a (46) 9b
5a 22a 24 a (24) 13b
la 9s 6\6) 19b
19 a 18 a (32)
31ab 41 a (42)
50s 51 a (47)
0" 3a 8 a (14) 0b
3b 14ab 28 a (29) 2a
3b 16* 35 a (32) 3a
0b 4 a (8)
6a 5 a (5)
10s 16 a (15)
15 b 38 a 56"(100)
23c 73b 98 a (100)
26b 85a 108 a (100)
(wk) 1 2 3
4" 19 s 19a(53)2
Upper beak
1 2 3
6" 14a 13a(36)
Lower beak
1 2 3
0" 0* 4 a (ll)
Tongue
1 2 3
0" 0" (PCO)
1 2 3
10b 33" 36 a (100)
Total mouth parts with lesions
a-c Values in a column within a lesion site with no common superscripts differ significantly (P<.05). Values are based on all birds fed the indicated toxin.
'TAS = triacetoxyscirpenol; STO = scirpentriol; MAS = monoacetoxyscirpenol; and D A S = diacetoxyscirpenol. t a b u l a r values in parentheses are the numbers of a particular mouth part having lesions expressed as the percentage of the total number of the mouth part exhibiting lesions after 3 wk exposure to the particular toxin. For example, TAS caused 36 total mouth parts to have lesions after 3 wk and 19 of them were in the angles o f the mouth, giving an incidence of 53% ([19 + 36] x 100) of the lesions occurring in the angles. Numbers are based on all birds receiving the toxin, i.e., the numbers for all treatment levels of the toxin were summed.
parts having lesions were considered, the MED were .5, 1.0, 2.0, and 4.0 u.g/g for MAS, DAS, STO, and TAS, respectively, giving an eightfold difference between MAS, the most effective, and TAS, the least effective (Table 3). When the different parts of the mouth were considered, there were no significant (P<.05) dose effects of TAS and STO on the lower beak or tongue nor of MAS on the angles. When the part of the mouth on which the toxins exhibited the greatest effect was considered, the MED of TAS and STO on the angles were 8.0 and 4.0 |Xg/g, respectively, and the MED of MAS and DAS on the upper beak were 2.0 and 1.0 u.g/g, respectively. When the dose-response relationships of the lesions were considered, with the number of affected birds being the response (Table 4), the pattern was the same as that obtained when total number of mouth parts having lesions was the response. The MED were .5, 1.0, 2.0, and 4.0 u.g/g for MAS, DAS, STO, and TAS, respectively, the same values obtained when the total number of mouth parts having lesions were considered. The fact that each dose of MAS gave a response (total number of affected mouth parts or birds) that differed significantly (P<.05)
from the responses of the other doses (Tables 3 and 4) permitted a meaningful linear regression of the MAS data. When Y = number of affected birds and X = log2 of MAS concentration, the equation was Y = 3.2 + 7. IX (r = .98). When Y = number of affected mouth parts, the equation became Y = -.8 + 19.8X (r = .99). DISCUSSION
The time of exposure required to provoke mouth lesions was clearly 1 wk or less for all four scirpenols (Table 2). The number of mouth parts having lesions about tripled from the end of the 1st wk until the end of the 2nd wk with each toxin, but only MAS caused a significant (P<.05) increase during the 3rd wk. These findings agree well with those of Wyatt et al. (1972b), who observed onset of T-2 induced lesions at about 1 wk, and with those of Chi and Mirocha (1978), who observed onset at 5 days. In contrast, Hoerr et al. (1982a) found onset to be between 2 and 4 days and Chi et al. (1977) found onset at 2 wk of exposure. Nevertheless, the bulk of the data supports the conclusion that exposure for only a few days results in mouth lesions.
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Angles
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ADEMOYERO AND HAMILTON TABLE 3. Dose-responses of mouth lesions in broilers fed scirpenolr
Lesion site Angles
Upper beak
Dose (Hg/g diet) 0 .5 1 2 4 8 0 .5 1 2 4 8
Lower beak
TAS
MAS
DAS
i'
0° 12 ab 24" lc jbc gab lla 13" 0* 1* 3a 0* 0*
0* 0* 2a 8a 0"
'6*'
'6"'
0* 0* 0s
2a la la
0°
0°
0° llb 28 a
jab 18a 27 a
0° 0°b
10*
5 3bc 21*
16a 32° 58 b 83*
6° 7° 37 b 64'ab 1051
8C
Total
23D
26 a 36a
2Qab 44 a
2bc
a e ~ Values in a column within a lesion site with no common superscripts differ significantly (P<05). Values are based on all birds fed each dose of each toxin for 3 wk. J TAS = triacetoxyscirpenol; STO = scirpentriol; MAS = monoacetoxyscirpenol; and DAS = diacetoxyscirpenol.
The current findings of small, if any, increases in the number of mouth parts with lesions between the 2nd and 3rd wk of exposure agree with the results of Chi et al. (1977), who found a constant percentage of affected birds from 3 to 9 wk exposure. The current findings do not agree with the findings of Hoerr et al. (1982a), who observed that the size and number of oral lesions increased through 14 days exposure and decreased thereafter, nor do the current findings agree with their conclusion that oral mucosa becomes resistant after 14 days of exposure. Also contrary to their conclusions are field observations that mouth lesion problems in broiler breeder flocks continue for months (P. B. Hamilton, unpublished data) and laboratory observations that mouth lesions induced by
DAS in broiler breeders continue until after DAS is removed from the feed (R. Sigmon, J. Brake, and P. B. Hamilton, Dept. of Poultry Science, N. C. State University, Raleigh, NC, unpublished data). The latter two observations, the current findings, and those of Chi et al. (1977), support a conclusion that mouth lesions will not disappear until the responsible mycotoxin or toxins are removed from the feed. The locations of the lesion sites in the mouth have practical implications, because the locations of lesion sites in field outbreaks are not randomly dispersed (P. B. Hamilton, unpublished data). None of the scirpenols caused lesions on only a single part of the mouth, but the lesions caused by TAS and STO were mainly on the angles of the mouth
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6* Tongue
STO
(Number of mouth parts with lesions) 0" 5* 2a c 0b 5a 0b 16a 11* a 5* 16 lla 13a 13a 16a 6° 5C b 23 b 2 15° a s 38 ab 31 10 32 a 48 a 13a 19 s
MOUTH LESIONS AND MYCOTOXtNS
2087
TABLE 4. Frequency of mouth lesions in chickens fed scirpenol mycotoxinr Dose (ug/g diet) 0 .5 1 2 4 8
TAS l/39 b ... 3/40b 4/40b 12/39a 15/39"
STO
MAS
DAS
(Number of birds with lesions/total number of birds) 4/40° 3/38e 2/40° ... 8/38d 3/40° 3/40° 21/39c 19/40b ll/40 b 25/38 b 24/40 ab 13/39ab 30/36a 32/39" 21/39" ^^ ._^
a_e Values in a column with no common superscripts differ significantly (P<.05). Values are affected birds/birds exposed to indicated toxin for 3 wk. TAS = triacetoxyscirpenol; STO = scirpentriol; MAS = monoacetoxyscirpenol; and DAS = diacetoxysciipenol.
In the current study, the MED and the dose levels of a particular toxin that produced significantly (P<.05) different responses were identical whether the number of affected birds (Table 4) or the number of affected mouth parts (Table 3) was the dependent variable. Because it is easier to count affected birds rather than affected mouth parts, counting affected birds would probably be the preferred way of quantitatively analyzing the ability of trichothecenes to cause mouth lesions. When the toxicities of the scirpenols were compared using the number of affected mouth parts (Table 3) or of affected birds (Table 4) to determine MED, the rank order from greatest to least toxicity was MAS, DAS, STO, and TAS. This was quite different from the rank order MAS, DAS, and STO, which were greater than TAS when body weight was the criterion of toxicity (Table 1). The ratios between the MED for body weight and mouth lesions were 4, 2, 1, and 2 for MAS, DAS, STO, and TAS, respectively, indicating that mouth lesions were of equal or greater sensitivity as an indicator of scirpenol toxicoses than was body weight. The same relationship was observed with T-2 toxin, which belongs to another family of trichothecene toxins (Wyatt et al, 1972a, 1973). The dose-response relationships of the toxins in the present study, except for MAS, cannot be described in detail because of the generally small number of doses having a significant (P<.05) effect on the response. However, MAS, which satisfied the criterion of each dose level producing a response significantly different (P<.05) from the other dose levels, yielded linear regression equations with r>.98. These results imply that the doseresponse relationship of MAS, at least, is
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and rarely on the tongue. The derivatives, MAS and DAS, caused their greatest effect on the upper beak, followed by the lower beak, and least effect on the tongue, although DAS showed a greater effect on the tongue than the other scirpenols. These findings support conclusions that concentration of lesions in different parts of the mouth in different outbreaks is not inconsistent with assigning the cause to toxicosis produced by Fusarium species and that the predominant location of the lesions can be useful in assigning the cause to specific mycotoxins. The present findings on lesion sites are not in total agreement with some earlier observations. Speers et al. (1977) found essentially an equal number of lesions on the angles, mandibles, and tongues of birds fed MAS and zearalenone. Chi and Mirocha (1978) found in birds fed DAS essentially equal percentages of upper beaks (100%) and tongues (90%) bearing lesions but the lower beaks and angles had about half those percentages. Hoerr et al. (1982a) offered no numbers but stated that all mouth parts were affected and that many lesions were near the salivary duct openings of birds fed DAS. A concentration of mouth lesions in and surrounding salivary duct openings has been seen in field outbreaks (P. B. Hamilton, unpublished data), but only a few such lesions were seen in the current study. Hoerr et al. (1982a) offered as one explanation for such lesions that T-2 toxin was excreted in the saliva, presumably after gastrointestinal absorption. Because birds intubated with DAS did not have mouth lesions (Hoerr et al., 1981, 1982b), their other explanation that toxincontaining feed becomes moistened with saliva and adheres to and around the duct seems more likely.
2088
ADEMOYERO AND HAMILTON
The discrepancies between the present study and the studies cited earlier appear to have several causes. It can be calculated from the data of Speers et al. (1977) that the moldy com they used as the source of MAS contained 5% zearalenone. This seems very unlikely. If a decimal error is assumed, giving a more likely .5% zearalenone, then their reported level of 50 ug MAS/g diet becomes 5 ug/g and their growth and lesion frequency data essentially agree with the present study. It must be mentioned that their corn was molded with Fusarium roseum 'Gibbosum', which thenlaboratory later reported to produce DAS and its two isomers, 3,4-DAS and 3,15-DAS, as well as MAS and zearalenone (Mirocha et al., 1985). A mixture of these scirpenols and zearalenone might well give the peculiar pattern of lesion locations observed by Speers et al. (1977), i.e., approximately equal numbers of lesions on each mouth part. Hoerr et al. (1982a), who found that the concentration of DAS did not influence lesion severity and that the number and size of lesions peaked at 14 days exposure and declined thereafter, added crystalline DAS to a small amount of casein as a carrier and combined it with a commercial broiler starter mash instead of dissolving the crystals in a solvent and adding the solution to the diet. It is difficult to disperse crystalline material evenly at parts per million levels in feed, and even if good dispersion is achieved,
segregation can occur readily. Vitamins and trace minerals usually are added to diets as premixes in order to avoid such problems. Inadequate dispersion and segregation might well account for the failure of Hoerr et al. (1982a) to obtain a dose-related response. In addition to these problems, Speers et al. (1977), Chi and Mirocha (1978), and Hoerr et al. (1982a) used small numbers of birds (5 to 20) per treatment and few treatment levels (one to two) and frequently were unable to analyze their data statistically. The ability of the four scirpenols differing in degree of acetylation to induce mouth lesions in a dose-related manner along with the prior reported ability of T-2 toxin (Wyatt et al, 1972a,b, 1973) imply that trichothecenes in general might cause mouth lesions and associated productivity losses in poultry. Certainly, the current findings support the contention that the finding of mouth lesions in birds is ample reason to commence a mold and mycotoxin control program in poultry operations or to examine an existing control program. ACKNOWLEDGMENTS
The authors thank Hunter Edwards and Nancy Bailey for technical assistance. The research was supported in part by Cambridge Products, Lie, Springfield, MO 68501. REFERENCES Ademoyero, A. A., and P. B. Hamilton, 1989. Influence of degree of acetylation of scirpenol mycotoxins on feed refusal by chickens. Poultry Sci. 68:854-855. Bruning, J. L., and B. L. Kintz, 1977. Computational Handbook of Statistics, 2nd ed. Scott, Foresman and Co. Glenview, IL. Chi, M. S., and C. J. Mirocha, 1978. Necrotic oral lesions in chickens fed diacetoxyscirpenol, T-2 toxin, and crotocin. Poultry Sci. 57:807-808. Chi, M S., C. J. Mirocha, H. J. Kurtz, G. Weaver, F. Bates, and W. Shimoda, 1977. Subacute toxicity of T-2 toxin in broiler chicks. Poultry Sci. 56:306-313. Hoerr, F. J., W. W. Carlton, and B. Yagen, 1981. Mycotoxicosis caused by a single dose of T-2 toxin or diacetoxyscirpenol in broiler chickens. Vet Pathol. 18:652-664. Hoerr, F. J., W. W. Carlton, B. Yagen, and A. Z. Joffe, 1982a. Mycotoxicosis caused by either T-2 toxin or diacetoxyscirpenol in the diet of broiler chickens. Fundam. Appl. Toxicol. 2:121-124. Hoerr, F. J., W. W. Carlton, B. Yagen, and A. Z. Joffe, 1982b. Mycotoxicosis produced in broiler chickens by multiple doses of either T-2 toxin or diacetoxyscirpenol. Avian Pathol. 11:369-383. Huff, W. E., R. B. Harvey, L. F. Kubena, and G. E. Rottinghaus, 1988. Toxic synergism between afla-
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linear, in agreement with data for T-2 toxin (Wyatt et al., 1972b). Previous studies on DAS and MAS in chickens are inadequate for comparing with our dose-response data. Chi and Mirocha (1978) observed 100% of the 10 birds fed 5 ug DAS/g diet to have mouth lesions coupled with growth depression of 25%. The present comparable values by interpolation were about 85 and 28%, respectively. Further comparison cannot be made because Chi and Mirocha (1978) used only the one level of DAS. Hoerr et al. (1982a), who studied two levels of DAS (4 and 16 ug/g), stated without offering data that the concentration of DAS did not influence mouth lesion severity. However, they did state that 16 but not 4 ug DAS/g diet inhibited growth significantly (P<.05), again without offering data. Speers et al. (1977) reported that 25 and 50 ug MAS/g diet inhibited growth 22 and 28%, respectively, and each caused five of six birds, to have mouth lesions. With 4 ug MAS/g, 27% growth inhibition was obtained and 30 of 36 birds had mouth lesions.
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13495. Appl. Enviroa Microbiol. 53:457-^59. Richardson, K. E., and P. B. Hamilton, 1990. Comparative toxicity of scirpentriol and its acetylated derivatives. Poultry Sci. 69:397-402. Richardson, K. E., G. E. Toney, C. A. Haney, and P. B. Hamilton, 1989. Occurrence of scirpentriol and its seven acetylated derivatives in culture extracts of Fusarium sambucinum NRRL 13495. J. Food Prot 52:871-876. SAS Institute, 1982. SAS® User's Guide. 1982 ed. SAS Institute, Inc., Cary, NC. Sigg, H. P., R. Mauli, E. Flury, and D. Hauser, 1965. Die Konstitution von Diacetoxyscirpenol. Helv. Chim. Acta 48:962-988. Speers, G. M, C. J. Mirocha, C. M. Christensen, and J. C. Behrens, 1977. Effects on laying hens of feeding corn invaded by two species of Fusarium and pure T-2 mycotoxin. Poultry Sci. 56:98-102. Wyatt, R. D., J. R. Harris, P. B. Hamilton, and H. R. Burmeister, 1972a. Possible field outbreaks of fusariotoxicosis in avians. Avian Dis. 16:1123-1129. Wyatt, R. D., B. A. Weeks, P. B. Hamilton, and H. R. Burmeister, 1972b. Severe oral lesions in chickens caused by ingestion of dietary fnsariotoxin T-2. Appl. Microbiol. 24:251-257. Wyatt, R. D., P. B. Hamilton, and H. R. Burmeister, 1973. The effects of T-2 toxin in broiler chickens. Poultry Sci. 52:1853-1859.
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toxin and T-2 toxin in broiler chickens. Poultry Sci. 67:1418-1423. Kubena, L. F., R. B. Harvey, W. E. Huff, D. E. Corrier, T. D. Phillips, and G. E. Rottinghaus, 1989a. Influence of ochratoxin (OA) and T-2 toxin singly and in combination on broiler chickens. Poultry Sci. 68: 867-872. Kubena, L. F., W. E. Huff, R. B. Harvey, T. D. Phillips, and G. E. Rottinghaus, 1989b. Individual and combined toxicity of deoxynivalenol and T-2 toxin in broiler chicks. Poultry Sci. 68:622-626. Kubena, L. F., R. B. Harvey, W. E. Huff, D. E. Corrier, T. D. Phillips, and G. E. Rottinghaus, 1990. Efficacy of a hydrated sodium calcium aluminosilicate to reduce the toxicity of aflatoxin and T-2 toxin. Poultry Sci. 69:1078-1086. Mirocha, C. J., R. J. Pawlosky, T. X. Zhu, and Y. W. Lee, 1985. Chemistry and biological activity of Fusarium roseum mycotoxins. Pages 291-307 in: Trichothecenes and Other Mycotoxins. J. Lacey, ed. John Wiley and Sons, New York, NY. Richardson, K. E., and P. B. Hamilton, 1987a. Preparation of 4,15-diacetoxyscirpenol from cultures of Fusarium sambucinum NRRL 13495. Appl. Environ. Microbiol. 53:460-462. Richardson, K. E., and P. B. Hamilton, 1987b. Preparation of scirpentriol and triacetoxyscirpenol in good yield from cultures of Fusarium sambucinum NRRL
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