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Association of Low Levels of Aflatoxin in Feed with Productivity Losses in Commercial Broiler Operations1,2,3
ENVIRONMENT AND HEALTH Association of Low Levels of Aflatoxin in Feed with Productivity Losses in Commercial Broiler Operations 1,2 ' 3 F. T. JONES, W. H. HAGLER, and P. B. HAMILTON Department of Poultry Science, North Carolina State University, Raleigh, North Carolina 27650 (Received for publication October 16, 1981)
1982 Poultry Science 61:861-868 INTRODUCTION The importance of aflatoxin and other mycotoxins to the poultry industry lies in economic losses that might result from impaired immunity (Michael et al, 1973; Pier and Heddleston, 1970; Thaxton et al, 1974), impaired kidney function (Tung et al, 1973), increased protein requirements (Smith et al., 1971), poor pigmentation (Tung and Hamilton, 1973), carcass condemnations (Warren and Hamilton, 1980), altered vitamin metabolism (Hamilton et al., 1974), leg problems (Huff et al., 1980), decreased egg production (Hamilton
'Paper Number 8033 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC. 2 The use of tradenames in this publication does not imply endorsement by the North Carolina Agricultural Research Service nor criticism of similar ones not mentioned. 3 A preliminary report of this article was presented at the 70th Annual Meeting of the Poultry Science Association, Vancouver, British Columbia, August 1981.
and Garlich, 1971), poor growth and efficiency of feed conversion, and increased mortality (Smith and Hamilton, 1970). These potentialities have been realized all too frequently (Smith and Hamilton, 1970; Tung and Hamilton, 1973: Hamilton, 1971, 1978; Smith et al, 1976; Huff et al, 1980; Warren and Hamilton, 1980). The source of mycotoxins has been anywhere from the grain fields prior to harvest to the feeders in front of the birds (Smith and Hamilton, 1970), and the concentration of aflatoxin associated with field problems has ranged from < 3 0 ppb (Hamilton, 1978) to 101,000 ppb (Hamilton, 1971) with the severity of the problems ranging from a 1% depression in growth rate to high mortality. For reasons of mycotoxin interactions and scale, it is difficult, if not impossible, to establish in the laboratory minimal effective doses and subclinical states of mycotoxicoses that are applicable in the field. The logical recourse, then, is to survey using established epidemiological principles. Thus, the objectives of this study were to determine whether aflatoxin could be as-
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ABSTRACT Six growers from each of five broiler companies were classified into equal numbers of good, mediocre, and poor growers based on a productivity index. Feed samples were collected weekly during the growout period of a flock from each grower and from the mill where the feed was produced. Samples of corn from which the feed was made were also collected. Aflatoxin, moisture contents, and bacterial, coliform, and fungal counts of the samples were determined. On the same weekly basis, the temperature and relative humidity of the chicken houses were measured and the age of the feed samples ascertained. Aflatoxin concentration in feed of good growers was 6.1 ppb with an 18.0% frequency of contamination while the values for poor growers were 14.0 ppb and 31.3%. Mean aflatoxin concentration in corn of 1.2 ppb increased to 8.8 ppb in feed from the farms. Optimum conditions for aflatoxin formation were 19 to 27 C, 79 to 89% relative humidity, and 10 to 13% moisture. Increasing age of feed was associated with low moisture and high aflatoxin. High aflatoxin was associated with high relative humidity but not with fungal, bacterial, or coliform counts. These data suggest that aflatoxin is produced during and after feed manufacture in apparently normal operations, that low levels of aflatoxin are associated with productivity losses in apparently healthy broilers, and that the aflatoxin formation is associated with high relative humidity in houses and with long residence time of the feed in the house. Because relative humidity and residence time in houses can be altered, aflatoxin formation might be limited by controlling these two factors. (Key words.- aflatoxin, broiler productivity, feed age, relative humidity, temperature, moisture, microbial counts)
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sociated with productivity in broiler operations which were not known to be suffering from aflatoxicosis, whether the aflatoxin was being formed after manufacture of the feed, and whether some simple factors could be correlated with the formation of aflatoxin. MATERIALS AND METHODS
Statistical Analyses. Data were evaluated using the multiple regression and Duncan's multiple range procedures of Barr et al. (1976). RESULTS The characteristics of the different classifications of growers are given in Table 1. There
TABLE 1. Productivity
parameters Productivity class
Parameter Birds marketed Age (days) Body weight (g) Feed conversion Livability (%) Condemnations (%) Grower payment (el/chick)
Good 122,052 a 52. 6 ± .8 1760 +41 2.13 ± .02 95.98 + .46 a 1.39 ± .22a 12.02 ± .62a
Mediocre lll,290b 51. 9± .7 1737 + 37 2.15 ± .02 95.61 + . 9 1 a 1.19 + .09 a 11.49 ± .50*
Poor 105,267 c 52. 8 + .6 1719 + 26 2.16+ .03 92.78 + 2.92 b 1.73 + .20 b 10.86+ .55b
a,b,c Values in a row with different superscripts differ significantly (P<.05). Values are means ± SEM. There were 10 growers per class.
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Grower Selection. Five broiler companies located in North Carolina participated in this study. Six growers who received their day-old birds within 1 week of each other were selected from each company. The growers were classified on the basis of a productivity index (mean market weight X 100/feed conversion) calculated using the data from all flocks grown the previous year. The grower classifications were good (index of 185 to 200), mediocre (170 to 184), and poor (155 to 169). Two growers from each company were chosen in each classification. Sample Collection. Feed samples were collected weekly from each grower during the grow-out period of a flock. A small, portable vacuum sampler (Model No. 9320/8970, Black and Decker Manufacturing Company, Towson, MD 21204) was used for collecting about 75 g of feed from every other feeder pan in the house or from approximately every 9 m in trough-type feeders. These portions from within a house were combined and mixed by pouring them on a sheet of clean plastic and rolling the sample by lifting alternate corners of the sheet. In addition, samples of feed and ingredients were collected weekly at the feed mill by company personnel who were instructed on proper sampling techniques. Ingredients other than corn were free of aflatoxin and are
not included in this report. One sample each of broiler starter, grower, and finisher were collected each sampling time from a delivery truck while it was being loaded. Single samples of each lot of corn shipments received during a sampling day were taken; consequently, the feed sampled on a given day did not contain the corn sampled the same day. The time between corn delivery and feed manufacture varied with the company but rarely exceeded 1 week. After collection, the samples were transported to the laboratory in plastic bags and frozen at —25 C until analyzed. Measurements. Temperature and humidity readings were obtained weekly from the approximate conter of each poultry house using a psychrometer (Model 566-2, Bendix Corporation, Environmental and Process Instrument Division, 1400 Taylor Avenue, Baltimore, MD 21204). The time each house was visited weekly was varied so that all measurements in a house were not made at the same hour of a day. Moisture content of the feed was determined gravimetrically after heating 10 ± .01 g in an oven at 100 ± 2 C for 18 to 20 hr. Aflatoxin was measured by Association of Official Analytical Chemists (AOAC) method 26.031 (Horowitz, 1980). Production and processing data were obtained from company records. Feed age was the difference between sampling date and last prior delivery date.
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TABLE 2. Relation of microbial counts and aflatoxin concentration of feed to productivity class Productivity class Good
Parameter Bacteria (log, 0 /g) c Coliforms (log 10 /g)d Fungi (log 10 /g) e Aflatoxin concentration (ppb) Aflatoxin frequency (% positive)
6.46 4.60 3.90 6.13
± ± ± ±
Mediocre 5.74 4.00 3.45 3.2 a
6.60 5.40 4.54 6.5
18.0 + 6.8 a
± 6.16 ± 5.16 ±4.42 ± 2.3a
22.1 ± 4 . 7 a
Poor 6.71 5.43 4.64 14.0 31.3
± 6.07 ± 5.18 + 4.49 ± 4.5b 6.1b
Bacterial counts were done on Trypticase Soy Agar (Difco). Coliform counts were done on MacConkey's Agar (Difco). Fungal counts were done on Saboraud's Dextrose Agar (Difco).
was no significant (P<.05) difference among classes in marketing age of birds, body weight, or feed conversion, although there was a trend for the growers classified as good to have heavier (P = .15), more efficient (P = .15) birds. During the period under study, the good growers marketed about 10% more birds than the mediocre growers who marketed about 5% more birds than the poor growers. The good and mediocre growers had significantly (P<.05) better livability (about 3%) and fewer condemnations (about .4%) than the poor growers. The most important difference among the classes of growers was in the payment the growers received per chick marketed. The good growers received about 5% more in payment than mediocre growers who received about 5% more than poor growers. The bacterial, coliform, and fungal counts did not differ significantly (P<.05) with the grower classification (Table 2), although for each type of microorganism its mean count was highest in feed from poor growers and lowest in feed from good growers. It should be men-
tioned that the standard errors in the counts were so high that almost tenfold differences were not significant. Perhaps most important in the present study, the concentration of aflatoxin found in the feed of poor growers was over twice that found in the feed of good growers, and the frequency of contamination of feed from the poor growers was almost twice that of good growers (Table 2). Categorically, these findings on relations of aflatoxin to grower classification agree with the relations of livability, condemnation, and grower payment to grower classification. Analyses (Table 3) of corn samples taken from the feed mill revealed a low concentration of aflatoxin (1.2 ppb) which increased about sevenfold (8.8 ppb) in feed taken from the poultry houses. The concentration (6.0 ppb) in feed sampled at the mill just after manufacture was intermediate (6.0 ppb) between the two values but not significantly different from the two values. There was no significant difference in frequency of contamination at the different
TABLE 3. Survey of corn and feed for aflatoxin
Item
Source
Corn Feed Feed
Mill Mill Farm
Samples (No.) 57 114 222
Values with different superscripts differ significantly (P<.05).
Aflatoxin (Ppb) 1.2 a 6.0 a b 8.8 b
Aflatoxin (% Positive) 21.1 19.3 23.4
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a ' b Values in a row with different superscripts differ significantly (P<.05). Values are mean ± SEM.
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TABLE 6. Relation of temperature of poultry bouses to aflatoxin in feed from those houses Temperature (C)
< 19 19-21 22-24 25-27 2 8 - 30 31 - 33 >33
Samples (No.)
1- 5 6 - 10 1 1 - 15 16-20
132 64 20 6
Aflatoxin (PP'b) 7.9 a 8.0 a 10.7 a 27.9 b
Aflatoxin (% Positive) 20.5 a 23.4 a 30.0 a 66.7 b
ab ' Values in a column with different scripts differ significantly (P<.05).
super-
TABLE 5. Moisture and aflatoxin content of feed Moisture range (%) 9.5 - 9.9 10.0- 10.4 10.5-10.9 11.0- 11.4 11.5- 11.9 12.0- 12.4 12.5 - 12.9 13.0- 13.4 13.5 - 13.9 14.0- 14.4 14.5- 14.9 15.0 and >
Samples (No.) 3 4 22 27 22 40 27 26 22 22 8 7
Aflatoxin (ppb) 0a .8a
4.6abc 10.9 e 24.5 f g 4.bcde 10.0cde 3.8ab 4.0ab 3.5 a b 6 1 abed 12.9 e
Aflatoxin (% Positive) 0a
25.0 C 31.8 C 25.9 C 34.8 C 25.0 C 32.lc 12.5 b 9 jab 9 jab 25.0 C 28.6 e
3. b C Q C
' ' ' ' Values in a column with different superscripts differ significantly (P<.05).
3.1 a 9.6bc 6.5 b 11.5 C 3.0 a 1.6a 0a
Aflatoxin (% Positive) 12.5 a 21.4 a b 27.3 b 26.9 b 17.0 a b 18.6 a b 0C
super-
TABLE 7. Relation of aflatoxin in feed to relative humidity in poultry house
(%)
Age of feed
16 14 22 52 53 43 3
Aflatoxin (ppb)
a,b,c.Values in a column with different scripts differ significantly (P<.05).
Relative humidity
TABLE 4. Relation of aflatoxin to age of feed
Samples (No.)
<60
60-69 70-79 80-89 90-99
Samples (No.) 34 72
66 28 3
Aflatoxin (ppb) 2.0 a 2.7 a 11. l b 5.6 a b l.la
Aflatoxin (% Positive) 8.8 a 15.5 a 28.8 b 32 l b 33.3 b
a ' b Values in a column with different superscripts differ significantly (P<.05).
nonrepresentative numbers. If the response curve is simplified on this assumption, aflatoxin concentration peaked between 11 and 13% moisture and the curve for frequency of contamination had a broader plateau between 10 and 13% moisture. The relation of temperature in the poultry houses to the aflatoxin found in feed from the houses is given in Table 6. Concentration and frequency of contamination of aflatoxin appeared to have a rather broad peak between 19 and 27 C, which agrees reasonably well with the optimum of 27 C reported for the production of aflatoxin by Aspergillus flavus (Christensen, 1975). The relation of aflatoxin in feed to the relative humidity of the house from which the feed came is given in Table 7. Aflatoxin concentration peaked (11.1 ppb) between 70 and 79% relative humidity, although it was not significantly (P<.05) different from the con-
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sources. None of the 394 feed and corn samples contained more than 200 ppb, and only three samples contained more than 100 ppb. The relation of time to aflatoxin content was studied further by dividing the age of feed at the farm into four categories and determining the aflatoxin concentration and contamination frequency for each age category (Table 4). As the time that the feed resided in the house increased, both aflatoxin concentration and frequency of contamination increased. When the feed samples from the houses were categorized on the basis of moisture content (Table 5), there was not a simple linear relationship between moisture and aflatoxin frequency and concentration. Rather, the relationship appeared sinusoidal because of the contributions made by the categories for the highest and lowest moisture contents contained
AFLATOXIN AND BROILER PRODUCTIVITY
DISCUSSION
The present survey showed a clear association between aflatoxin concentration in feed and the productivity of flocks consuming the feed. Feed from houses of growers classed as good had a contamination frequency of 18.0% with a mean of 6.1 ppb while feed from houses of growers classed as poor had a frequency of 31.3% and a mean of 14.0 ppb. It should be noted that these levels of aflatoxin are very low, even lower than the old FDA guideline of 20 ppb and much lower than the minimal growth inhibitory concentration of 2500 ppb for young broilers in a widely used experimental model for aflatoxicosis in chickens (Smith and Hamilton, 1970). However, these field values are in reasonable agreement with other field observations such as retarded growth and feed conversion with <30 ppb and a frequency of 30% (Hamilton, 1978), clinical aflatoxicosis with 60 ppb (Smith et al, 1976), and poor growth, feed conversion, and pigmentation with <50 ppb (Hamilton, 1975). The apparent discrepancy between levels effective under field conditions and levels
effective in the laboratory may be explained by interactions occurring under field conditions between aflatoxin and numerous other undesirable factors such as inadequate nutrients (Smith et al, 1971), infectious agents (Wyatt and Hamilton, 1975), environmental extremes (Hamilton and Harris, 1971), and other mycotoxins (Lillehoj et al, 1975). Regardless of the reason, there are now several independent studies showing productivity losses in apparently healthy flocks associated with very low levels of aflatoxin (Smith and Hamilton, 1970; Smith et al, 1976; Hamilton, 1978). Clearly, efforts to establish "safe" levels of mycotoxins are going to require field studies with large numbers of birds, and they will include economic criteria in addition to the clinical criteria traditionally used in laboratory studies. The question of where and when the aflatoxin is formed is of equal importance. Since all flocks in this study were started the same week, they were subject to similar climatic conditions. All growers within a given company presumably have an equal chance of receiving a given lot of feed, hence, the finding of higher concentrations and frequency of aflatoxin in feed from poor growers suggests that aflatoxin was formed in the feed subsequent to delivery at the farm. This suggestion is supported by the increase in aflatoxin concentration and frequency as the feed age increases (Table 4). In fairness, it should be mentioned that while the aflatoxin concentration and frequency increased from mill samples to farm samples (Table 3), The increase was not significant (P<.05). This apparent discrepancy resides in the age distribution of the farm samples (Table 4) being dominated by samples of short residence time; therefore, the age effect is diluted out in an overall analysis (Table 3, Table 8). The validity of the feed age effect noted here is supported by the observation of Good and Hamilton (1981) that shortening the residence time of feed in a house relieved moldy feed problems. The evidence also suggests that aflatoxin was formed at the mill, because corn which is the ingredient supporting toxin formation contained only 1.2 ppb while the feed leaving the mills had a mean aflatoxin concentration of 6.0 ppb (Table 3). An earlier study (Smith and Hamilton, 1970) found aflatoxin formation at both the mill and farm levels as did a study on
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centration (5.6 ppb) occurring in samples taken between 80 and 89% relative humidity. A different pattern occurred with frequency of contamination, which had a broad plateau (about 30% of the samples being contaminated between 70 and 99% relative humidity). The lack of better correspondence between concentration and frequency of contamination may reside in the small number of samples in the 80 to 89% category (N = 28) and 90 to 99% category (N = 3). The relationship between the various factors measured in this study were evaluated in another way with a Pearson product-moment correlation analysis in which all possible comparisons were made (Table 8). Of the 45 correlations based on a minimum of 200 observations per comparison only 7 were significant (P<.05): high aflatoxin concentration and low grower classification, high temperature and low moisture, long feed age and low moisture, high bacterial count and high coliform-count, high bacterial count and high fungal count, low temperature and high fungal count, and low temperature and high relative humidity. The correlation between high relative humidity and high aflatoxin concentration was P = .069.
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b
1.000* 0b 222 c
Number of observations.
Probability >(R).
Correlation coefficient.
Feed age
Relative humidity
Temperature
Aflatoxin concentration
Fungi
Coliforms
Bacteria
Moisture
Grow class
Grow class
.004 .950 222 1.000 0 222
Moisture
222 0 222
1.000
222
0
.094 .168 219 219 -.026 .706 219 219 .143 .029
Coliforms
.096 .158 219 219 .061 .372 219 219 1.000
Bacteria .073 .282 219 .013 .854 219 .449 .001 222 .049 .463 222 1.000 0 222 0 222
1.000
.025 "'" .718 2219 19
219
-.016 .809
219
.002 .977
222
.050 .460
222
.129 .050
Aflatoxin concentration
coefficients
Fungi
TABLE 8. Correlation
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AFLATOXIN AND BROILER PRODUCTIVITY
Schroeder (1969) stated that the three most important factors influencing aflatoxin formation in stored field crops were moisture,
relative humidity, and temperature. The current data suggest these factors have a similar influence in poultry feed, although they differ in detail. The optimum conditions for production of aflatoxin in broiler feed appeared to be 10 to 13% moisture and 70 to 89% relative humidity which are lower than those reported for stored grains (Christensen, 1975), and 19 to 27 C, which agrees with laboratory findings* of Schroeder and Hein (1967). The greatest difference noted in the present study from prior reports would appear to be in the low moisture level which is lower than the average moisture level in poultry feed manufactured in North Carolina (Tabib et al., 1981), associated here with peak aflatoxin production. The intimate relationship between temperature, moisture, and relative humidity can be used to reconcile the current data. Once the feed in this study entered the house, it started dessicating, as indicated by the correlations between high temperature and low moisture, between long feed age and low moisture, and between high temperature and low relative humidity (Table 8). A correlation between high relative humidity and high aflatoxin concentration rather than between moisture and aflatoxin suggests that the microenvironment supporting aflatoxin production is near the feed surface or it would not be influenced in such a fashion in feed of low moisture content. The early formation of aflatoxin prior to dessication does not seem likely because of the relationship between feed age and aflatoxin concentration (Table 4). Instead, there must be some mechanism that accounts for a moisture optimum of below 13% while the optimal relative humidity is in equilibrium with corn of 16 to 18% moisture (Christensen and Meronuck, 1974). The major difficulty with invoking microenvironments near the feed surface as the site of aflatoxin formation is how to account for dessication in an atmosphere of high relative humidity. This can be done by assuming gradients of water vapor in the house, including the feed, on a permanent or cyclical basis, for example, distillation of water from the feed bin coupled with high relative humidity in the house because of inadequate ventilation. We emphasize that aflatoxin does not occur uniformly throughout feed. Overall, only about 22% of the samples contained detectable aflatoxin and only about 31% of the samples from the poor class of growers were positive. The concentration of aflatoxin in individual
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ochratoxin (Hamilton, Huff, Harris, and Wyatt, unpublished results), while studies such as Stepehnson (1974), which looked only at the farm level, found toxin formation there. These results and conclusions appear to conflict with opinions based on model studies that fungal growth and the production of mycotoxins in stored grains and products is unlikely (Christensen, 1980). The current results have several microbiological implications. The failure of fungal counts to correlate significantly with aflatoxin concentration in feed samples can be explained since only the A. flavus-parasiticus group of the nearly 15,000 species in the Fungi Imperfecti produce aflatoxin (Landecker, 1972), and the medium used to obtain the fungal count was not selective. Thus, it is not likely that measurement of total fungal load would detect shifts within the fungal population. In addition, Tuite (1979) suggested toxic fungi may grow vigorously without producing toxin because the mycotoxin is metabolized, competing fungi use essential nutrients, the environment does not permit toxin formation, and nontoxic strains predominate over toxic strains. The correlation of fungal counts with bacterial counts in a rather dry environment such as feed would appear to be contradictory, since bacterial growth and activity require free water (i.e., free to migrate in capillary spaces as opposed to bound water, which may or may not be available for bacterial growth (Kramer, 1969). The correlation of bacterial counts with coliform counts in feed is also surprising, because coliforms are thought to inhabit aqueous environments. However, another study (Tabib et al., 1981) found increased numbers of coliforms and fungi in feed of suspect microbiological quality. This finding of the present study suggests that the microbial activity under discussion does not occur in the average environment measured but rather that the microorganisms grow in microenvironments not representative of the whole, yet permitting microbial growth commonly associated with the more favorable available water relationships seen in the laboratory. Majumder et al. (1965) showed that moisture migration in sealed containers of grain as a result of temperature gradients encouraged microbial growth in the absence of a net gain in moisture.
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samples ranged from 0 to 196 ppb. The folly of using single samples to assess a possible mycotoxin problem would seem obvious. Trying to duplicate field exposure of animals to aflatoxin in the laboratory would seem to be a formidable task. ACKNOWLEDGMENTS
The technical assistance of D. Joy Osborne and Jim Hutchins is greatly appreciated.
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REFERENCES Barr, A. J., J. H. Goodnight, J. P. Sail, and J. T. Helwig, 1976. A User's Guide to SAS 76. SAS Inst., Inc., Raleigh, NC. Christensen, C. M., 1975. Molds, Mushrooms and Mycotoxins. Univ. Minnesota Press, Minneapolis, MN. Christensen, C. M., 1980. Needed: Research on storage molds in grains, seeds and their products. Plant Dis. 64:1067-1070. .Christensen, C. M., and R. A. Meronuck, 1974. Manual of Fungi in Feeds, Foods, and Cereal Grains. Univ. Minn. Agric. Ext. Serv., St. Paul, MN. Good, R. E., and P. B. Hamilton, 1981. Beneficial effect of reducing the feed residence time in a field problem of suspected moldy feed. Poultry Sci. 60:1403-1405. Hamilton, P. B., 1971. A natural and extremely severe occurrence of aflatoxicosis in laying hens. Poultry Sci. 50:1880-1882. Hamilton, P. B., 1975. Proof of mycotoxicosis being a field problem and a simple method for their control. Poultry Sci. 54:1706-1708. Hamilton, P. B., 1978. Fallacies in our understanding of mycotoxins. J. Food Prot. 41:404—408. Hamilton, P. B., and J. D. Garlich, 1971. Aflatoxin as a possible cause of fatty liver syndrome in laying hens. Poultry Sci. 50:800-804. Hamilton, P. B., and J. R. Harris, 1971. Interaction of aflatoxicosis with Candida albicans infections and other stresses in the chicken. Poultry Sci. 50: 906-912. Hamilton, P. B., H. T. Tung, R. D. Wyatt, and W. E. Donaldson, 1974. Interaction of dietary aflatoxin with some vitamin deficiencies. Poultry Sci. 53:871-877. Horowitz, W., ed., 1980. Methods of Analysis of the Association of Official Analytical Chemists. Assoc. Offic. Anal. Chem., Washington, DC. Huff, W. E., J. A. Doerr, P. B. Hamilton, D. D. Hainason, R. E. Peterson, and A. Ciegler, 1980. Evaluation of bone strength during aflatoxicosis and ochratoxicosis. Appl. Environ. Microbiol. 40: 102-107. Kramer, P. J., 1969. Plant and Soil Water Relationships: A Modern Synthesis. McGraw Hill Book Co., New York, NY. Landecker, E. M., 1972. Fundamentals of the Fungi.
Prentice-Hall, Inc. Englewood Cliffs, NJ. Lillehoj, E. B., and A. Ciegler, 1975. Mycotoxin Synergism. In Microbiology - 1975. D. Schlessinger, ed. Am. Soc. Microbiol., Washington, DC. Majumder, S. K., K. S. Narasimhan, and H.A.B. Parpia, 1965. Microecological factors of microbial spoilage and the occurrence of mycotoxins on stored grains Pages 2 7 - 4 7 in Mycotoxins in Foodstuffs. G. N. Wogan, ed. MIT Press. Amherst, MA. Michael, G. Y., P. Thaxton, and P. B. Hamilton, 1973. Impairment of the reticuloendothelial system of chickens during aflatoxicosis. Poultry Sci. 52: 1206-1207. Pier, A. C , and K. L. Huddleston, 1970. The effect of aflatoxin in immunity in turkeys. I. Impairment of actively acquired resistance to bacterial challenge. Avian Dis. 14:797-809. Schroeder, H. W., 1969. Factors influencing the development of aflatoxins in some field crops. J. Stored Prod. Res. 5:187-192. Schroeder, H. W., and H. Hein, 1967. Aflatoxins: Production of the toxins In vitro in relation to temperature. Appl. Microbiol. 15:441—445. Smith, J. W., and P. B. Hamilton, 1970. Aflatoxicosis in the broiler chicken. Poultry Sci. 49:207-215. Smith, J. W., C. H. Hill, and W. E. Donaldson, 1971. The effect of dietary modifications on aflatoxicosis in the broiler chicken. Poultry Sci. 50:768-774. Smith, R. B., J. H. Griffin, and P. B. Hamilton, 1976. Survey of aflatoxicosis in farm animals. Appl. Environ. Microbiol. 31:385-388. Stephenson, E. L., 1974. The relation of mold to bone disorders in poultry. Proc. North Carolina Poultry Nutr. Conf. 1:1-2. Tabib, Z., F. T. Jones, and P. B. Hamilton, 1981. Microbiological quality of poultry feed and ingredients. Poultry Sci 60:1392-1397. Thaxton, P., H. T. Tung, and P. B. Hamilton, 1974. Immunosuppression in chickens by aflatoxin. Poultry Sci. 53:721-725. Tuite, J., 1979. Field storage conditions for the production of mycotoxins and geographic distributions of some mycotoxins in problems in the United States. In: Interactions of Mycotoxins in Animal Production. Natl. Acad. Sci., Washington, DC. Tung, H. T., and P. B. Hamilton, 1973. Decreased plasma carotenoids during aflatoxicosis. Poultry Sci. 5 2 : 8 0 - 8 3 . Tung, H. T., R. D. Wyatt, P. Thaxton, and P. B. Hamilton. 1973. Impairment of kidney function during aflatoxicosis. Poultry Sci. 52:873-878. Warren, M. F., and P. B. Hamilton, 1980. Intestinal fragility during ochratoxicosis and aflatoxicosis in broiler chickens. Appl. Environ. Microbiol. 40:641-645. Wyatt, R. D., and P. B. Hamilton, 1975. Interaction between aflatoxicosis and a natural infection of chickens with Salmonella. Appl. Microbiol. 30:870-872.
1982 Poultry Science 61:861-868 INTRODUCTION The importance of aflatoxin and other mycotoxins to the poultry industry lies in economic losses that might result from impaired immunity (Michael et al, 1973; Pier and Heddleston, 1970; Thaxton et al, 1974), impaired kidney function (Tung et al, 1973), increased protein requirements (Smith et al., 1971), poor pigmentation (Tung and Hamilton, 1973), carcass condemnations (Warren and Hamilton, 1980), altered vitamin metabolism (Hamilton et al., 1974), leg problems (Huff et al., 1980), decreased egg production (Hamilton
'Paper Number 8033 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC. 2 The use of tradenames in this publication does not imply endorsement by the North Carolina Agricultural Research Service nor criticism of similar ones not mentioned. 3 A preliminary report of this article was presented at the 70th Annual Meeting of the Poultry Science Association, Vancouver, British Columbia, August 1981.
and Garlich, 1971), poor growth and efficiency of feed conversion, and increased mortality (Smith and Hamilton, 1970). These potentialities have been realized all too frequently (Smith and Hamilton, 1970; Tung and Hamilton, 1973: Hamilton, 1971, 1978; Smith et al, 1976; Huff et al, 1980; Warren and Hamilton, 1980). The source of mycotoxins has been anywhere from the grain fields prior to harvest to the feeders in front of the birds (Smith and Hamilton, 1970), and the concentration of aflatoxin associated with field problems has ranged from < 3 0 ppb (Hamilton, 1978) to 101,000 ppb (Hamilton, 1971) with the severity of the problems ranging from a 1% depression in growth rate to high mortality. For reasons of mycotoxin interactions and scale, it is difficult, if not impossible, to establish in the laboratory minimal effective doses and subclinical states of mycotoxicoses that are applicable in the field. The logical recourse, then, is to survey using established epidemiological principles. Thus, the objectives of this study were to determine whether aflatoxin could be as-
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ABSTRACT Six growers from each of five broiler companies were classified into equal numbers of good, mediocre, and poor growers based on a productivity index. Feed samples were collected weekly during the growout period of a flock from each grower and from the mill where the feed was produced. Samples of corn from which the feed was made were also collected. Aflatoxin, moisture contents, and bacterial, coliform, and fungal counts of the samples were determined. On the same weekly basis, the temperature and relative humidity of the chicken houses were measured and the age of the feed samples ascertained. Aflatoxin concentration in feed of good growers was 6.1 ppb with an 18.0% frequency of contamination while the values for poor growers were 14.0 ppb and 31.3%. Mean aflatoxin concentration in corn of 1.2 ppb increased to 8.8 ppb in feed from the farms. Optimum conditions for aflatoxin formation were 19 to 27 C, 79 to 89% relative humidity, and 10 to 13% moisture. Increasing age of feed was associated with low moisture and high aflatoxin. High aflatoxin was associated with high relative humidity but not with fungal, bacterial, or coliform counts. These data suggest that aflatoxin is produced during and after feed manufacture in apparently normal operations, that low levels of aflatoxin are associated with productivity losses in apparently healthy broilers, and that the aflatoxin formation is associated with high relative humidity in houses and with long residence time of the feed in the house. Because relative humidity and residence time in houses can be altered, aflatoxin formation might be limited by controlling these two factors. (Key words.- aflatoxin, broiler productivity, feed age, relative humidity, temperature, moisture, microbial counts)
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sociated with productivity in broiler operations which were not known to be suffering from aflatoxicosis, whether the aflatoxin was being formed after manufacture of the feed, and whether some simple factors could be correlated with the formation of aflatoxin. MATERIALS AND METHODS
Statistical Analyses. Data were evaluated using the multiple regression and Duncan's multiple range procedures of Barr et al. (1976). RESULTS The characteristics of the different classifications of growers are given in Table 1. There
TABLE 1. Productivity
parameters Productivity class
Parameter Birds marketed Age (days) Body weight (g) Feed conversion Livability (%) Condemnations (%) Grower payment (el/chick)
Good 122,052 a 52. 6 ± .8 1760 +41 2.13 ± .02 95.98 + .46 a 1.39 ± .22a 12.02 ± .62a
Mediocre lll,290b 51. 9± .7 1737 + 37 2.15 ± .02 95.61 + . 9 1 a 1.19 + .09 a 11.49 ± .50*
Poor 105,267 c 52. 8 + .6 1719 + 26 2.16+ .03 92.78 + 2.92 b 1.73 + .20 b 10.86+ .55b
a,b,c Values in a row with different superscripts differ significantly (P<.05). Values are means ± SEM. There were 10 growers per class.
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Grower Selection. Five broiler companies located in North Carolina participated in this study. Six growers who received their day-old birds within 1 week of each other were selected from each company. The growers were classified on the basis of a productivity index (mean market weight X 100/feed conversion) calculated using the data from all flocks grown the previous year. The grower classifications were good (index of 185 to 200), mediocre (170 to 184), and poor (155 to 169). Two growers from each company were chosen in each classification. Sample Collection. Feed samples were collected weekly from each grower during the grow-out period of a flock. A small, portable vacuum sampler (Model No. 9320/8970, Black and Decker Manufacturing Company, Towson, MD 21204) was used for collecting about 75 g of feed from every other feeder pan in the house or from approximately every 9 m in trough-type feeders. These portions from within a house were combined and mixed by pouring them on a sheet of clean plastic and rolling the sample by lifting alternate corners of the sheet. In addition, samples of feed and ingredients were collected weekly at the feed mill by company personnel who were instructed on proper sampling techniques. Ingredients other than corn were free of aflatoxin and are
not included in this report. One sample each of broiler starter, grower, and finisher were collected each sampling time from a delivery truck while it was being loaded. Single samples of each lot of corn shipments received during a sampling day were taken; consequently, the feed sampled on a given day did not contain the corn sampled the same day. The time between corn delivery and feed manufacture varied with the company but rarely exceeded 1 week. After collection, the samples were transported to the laboratory in plastic bags and frozen at —25 C until analyzed. Measurements. Temperature and humidity readings were obtained weekly from the approximate conter of each poultry house using a psychrometer (Model 566-2, Bendix Corporation, Environmental and Process Instrument Division, 1400 Taylor Avenue, Baltimore, MD 21204). The time each house was visited weekly was varied so that all measurements in a house were not made at the same hour of a day. Moisture content of the feed was determined gravimetrically after heating 10 ± .01 g in an oven at 100 ± 2 C for 18 to 20 hr. Aflatoxin was measured by Association of Official Analytical Chemists (AOAC) method 26.031 (Horowitz, 1980). Production and processing data were obtained from company records. Feed age was the difference between sampling date and last prior delivery date.
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863
TABLE 2. Relation of microbial counts and aflatoxin concentration of feed to productivity class Productivity class Good
Parameter Bacteria (log, 0 /g) c Coliforms (log 10 /g)d Fungi (log 10 /g) e Aflatoxin concentration (ppb) Aflatoxin frequency (% positive)
6.46 4.60 3.90 6.13
± ± ± ±
Mediocre 5.74 4.00 3.45 3.2 a
6.60 5.40 4.54 6.5
18.0 + 6.8 a
± 6.16 ± 5.16 ±4.42 ± 2.3a
22.1 ± 4 . 7 a
Poor 6.71 5.43 4.64 14.0 31.3
± 6.07 ± 5.18 + 4.49 ± 4.5b 6.1b
Bacterial counts were done on Trypticase Soy Agar (Difco). Coliform counts were done on MacConkey's Agar (Difco). Fungal counts were done on Saboraud's Dextrose Agar (Difco).
was no significant (P<.05) difference among classes in marketing age of birds, body weight, or feed conversion, although there was a trend for the growers classified as good to have heavier (P = .15), more efficient (P = .15) birds. During the period under study, the good growers marketed about 10% more birds than the mediocre growers who marketed about 5% more birds than the poor growers. The good and mediocre growers had significantly (P<.05) better livability (about 3%) and fewer condemnations (about .4%) than the poor growers. The most important difference among the classes of growers was in the payment the growers received per chick marketed. The good growers received about 5% more in payment than mediocre growers who received about 5% more than poor growers. The bacterial, coliform, and fungal counts did not differ significantly (P<.05) with the grower classification (Table 2), although for each type of microorganism its mean count was highest in feed from poor growers and lowest in feed from good growers. It should be men-
tioned that the standard errors in the counts were so high that almost tenfold differences were not significant. Perhaps most important in the present study, the concentration of aflatoxin found in the feed of poor growers was over twice that found in the feed of good growers, and the frequency of contamination of feed from the poor growers was almost twice that of good growers (Table 2). Categorically, these findings on relations of aflatoxin to grower classification agree with the relations of livability, condemnation, and grower payment to grower classification. Analyses (Table 3) of corn samples taken from the feed mill revealed a low concentration of aflatoxin (1.2 ppb) which increased about sevenfold (8.8 ppb) in feed taken from the poultry houses. The concentration (6.0 ppb) in feed sampled at the mill just after manufacture was intermediate (6.0 ppb) between the two values but not significantly different from the two values. There was no significant difference in frequency of contamination at the different
TABLE 3. Survey of corn and feed for aflatoxin
Item
Source
Corn Feed Feed
Mill Mill Farm
Samples (No.) 57 114 222
Values with different superscripts differ significantly (P<.05).
Aflatoxin (Ppb) 1.2 a 6.0 a b 8.8 b
Aflatoxin (% Positive) 21.1 19.3 23.4
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a ' b Values in a row with different superscripts differ significantly (P<.05). Values are mean ± SEM.
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TABLE 6. Relation of temperature of poultry bouses to aflatoxin in feed from those houses Temperature (C)
< 19 19-21 22-24 25-27 2 8 - 30 31 - 33 >33
Samples (No.)
1- 5 6 - 10 1 1 - 15 16-20
132 64 20 6
Aflatoxin (PP'b) 7.9 a 8.0 a 10.7 a 27.9 b
Aflatoxin (% Positive) 20.5 a 23.4 a 30.0 a 66.7 b
ab ' Values in a column with different scripts differ significantly (P<.05).
super-
TABLE 5. Moisture and aflatoxin content of feed Moisture range (%) 9.5 - 9.9 10.0- 10.4 10.5-10.9 11.0- 11.4 11.5- 11.9 12.0- 12.4 12.5 - 12.9 13.0- 13.4 13.5 - 13.9 14.0- 14.4 14.5- 14.9 15.0 and >
Samples (No.) 3 4 22 27 22 40 27 26 22 22 8 7
Aflatoxin (ppb) 0a .8a
4.6abc 10.9 e 24.5 f g 4.bcde 10.0cde 3.8ab 4.0ab 3.5 a b 6 1 abed 12.9 e
Aflatoxin (% Positive) 0a
25.0 C 31.8 C 25.9 C 34.8 C 25.0 C 32.lc 12.5 b 9 jab 9 jab 25.0 C 28.6 e
3. b C Q C
' ' ' ' Values in a column with different superscripts differ significantly (P<.05).
3.1 a 9.6bc 6.5 b 11.5 C 3.0 a 1.6a 0a
Aflatoxin (% Positive) 12.5 a 21.4 a b 27.3 b 26.9 b 17.0 a b 18.6 a b 0C
super-
TABLE 7. Relation of aflatoxin in feed to relative humidity in poultry house
(%)
Age of feed
16 14 22 52 53 43 3
Aflatoxin (ppb)
a,b,c.Values in a column with different scripts differ significantly (P<.05).
Relative humidity
TABLE 4. Relation of aflatoxin to age of feed
Samples (No.)
<60
60-69 70-79 80-89 90-99
Samples (No.) 34 72
66 28 3
Aflatoxin (ppb) 2.0 a 2.7 a 11. l b 5.6 a b l.la
Aflatoxin (% Positive) 8.8 a 15.5 a 28.8 b 32 l b 33.3 b
a ' b Values in a column with different superscripts differ significantly (P<.05).
nonrepresentative numbers. If the response curve is simplified on this assumption, aflatoxin concentration peaked between 11 and 13% moisture and the curve for frequency of contamination had a broader plateau between 10 and 13% moisture. The relation of temperature in the poultry houses to the aflatoxin found in feed from the houses is given in Table 6. Concentration and frequency of contamination of aflatoxin appeared to have a rather broad peak between 19 and 27 C, which agrees reasonably well with the optimum of 27 C reported for the production of aflatoxin by Aspergillus flavus (Christensen, 1975). The relation of aflatoxin in feed to the relative humidity of the house from which the feed came is given in Table 7. Aflatoxin concentration peaked (11.1 ppb) between 70 and 79% relative humidity, although it was not significantly (P<.05) different from the con-
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sources. None of the 394 feed and corn samples contained more than 200 ppb, and only three samples contained more than 100 ppb. The relation of time to aflatoxin content was studied further by dividing the age of feed at the farm into four categories and determining the aflatoxin concentration and contamination frequency for each age category (Table 4). As the time that the feed resided in the house increased, both aflatoxin concentration and frequency of contamination increased. When the feed samples from the houses were categorized on the basis of moisture content (Table 5), there was not a simple linear relationship between moisture and aflatoxin frequency and concentration. Rather, the relationship appeared sinusoidal because of the contributions made by the categories for the highest and lowest moisture contents contained
AFLATOXIN AND BROILER PRODUCTIVITY
DISCUSSION
The present survey showed a clear association between aflatoxin concentration in feed and the productivity of flocks consuming the feed. Feed from houses of growers classed as good had a contamination frequency of 18.0% with a mean of 6.1 ppb while feed from houses of growers classed as poor had a frequency of 31.3% and a mean of 14.0 ppb. It should be noted that these levels of aflatoxin are very low, even lower than the old FDA guideline of 20 ppb and much lower than the minimal growth inhibitory concentration of 2500 ppb for young broilers in a widely used experimental model for aflatoxicosis in chickens (Smith and Hamilton, 1970). However, these field values are in reasonable agreement with other field observations such as retarded growth and feed conversion with <30 ppb and a frequency of 30% (Hamilton, 1978), clinical aflatoxicosis with 60 ppb (Smith et al, 1976), and poor growth, feed conversion, and pigmentation with <50 ppb (Hamilton, 1975). The apparent discrepancy between levels effective under field conditions and levels
effective in the laboratory may be explained by interactions occurring under field conditions between aflatoxin and numerous other undesirable factors such as inadequate nutrients (Smith et al, 1971), infectious agents (Wyatt and Hamilton, 1975), environmental extremes (Hamilton and Harris, 1971), and other mycotoxins (Lillehoj et al, 1975). Regardless of the reason, there are now several independent studies showing productivity losses in apparently healthy flocks associated with very low levels of aflatoxin (Smith and Hamilton, 1970; Smith et al, 1976; Hamilton, 1978). Clearly, efforts to establish "safe" levels of mycotoxins are going to require field studies with large numbers of birds, and they will include economic criteria in addition to the clinical criteria traditionally used in laboratory studies. The question of where and when the aflatoxin is formed is of equal importance. Since all flocks in this study were started the same week, they were subject to similar climatic conditions. All growers within a given company presumably have an equal chance of receiving a given lot of feed, hence, the finding of higher concentrations and frequency of aflatoxin in feed from poor growers suggests that aflatoxin was formed in the feed subsequent to delivery at the farm. This suggestion is supported by the increase in aflatoxin concentration and frequency as the feed age increases (Table 4). In fairness, it should be mentioned that while the aflatoxin concentration and frequency increased from mill samples to farm samples (Table 3), The increase was not significant (P<.05). This apparent discrepancy resides in the age distribution of the farm samples (Table 4) being dominated by samples of short residence time; therefore, the age effect is diluted out in an overall analysis (Table 3, Table 8). The validity of the feed age effect noted here is supported by the observation of Good and Hamilton (1981) that shortening the residence time of feed in a house relieved moldy feed problems. The evidence also suggests that aflatoxin was formed at the mill, because corn which is the ingredient supporting toxin formation contained only 1.2 ppb while the feed leaving the mills had a mean aflatoxin concentration of 6.0 ppb (Table 3). An earlier study (Smith and Hamilton, 1970) found aflatoxin formation at both the mill and farm levels as did a study on
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centration (5.6 ppb) occurring in samples taken between 80 and 89% relative humidity. A different pattern occurred with frequency of contamination, which had a broad plateau (about 30% of the samples being contaminated between 70 and 99% relative humidity). The lack of better correspondence between concentration and frequency of contamination may reside in the small number of samples in the 80 to 89% category (N = 28) and 90 to 99% category (N = 3). The relationship between the various factors measured in this study were evaluated in another way with a Pearson product-moment correlation analysis in which all possible comparisons were made (Table 8). Of the 45 correlations based on a minimum of 200 observations per comparison only 7 were significant (P<.05): high aflatoxin concentration and low grower classification, high temperature and low moisture, long feed age and low moisture, high bacterial count and high coliform-count, high bacterial count and high fungal count, low temperature and high fungal count, and low temperature and high relative humidity. The correlation between high relative humidity and high aflatoxin concentration was P = .069.
865
b
1.000* 0b 222 c
Number of observations.
Probability >(R).
Correlation coefficient.
Feed age
Relative humidity
Temperature
Aflatoxin concentration
Fungi
Coliforms
Bacteria
Moisture
Grow class
Grow class
.004 .950 222 1.000 0 222
Moisture
222 0 222
1.000
222
0
.094 .168 219 219 -.026 .706 219 219 .143 .029
Coliforms
.096 .158 219 219 .061 .372 219 219 1.000
Bacteria .073 .282 219 .013 .854 219 .449 .001 222 .049 .463 222 1.000 0 222 0 222
1.000
.025 "'" .718 2219 19
219
-.016 .809
219
.002 .977
222
.050 .460
222
.129 .050
Aflatoxin concentration
coefficients
Fungi
TABLE 8. Correlation
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AFLATOXIN AND BROILER PRODUCTIVITY
Schroeder (1969) stated that the three most important factors influencing aflatoxin formation in stored field crops were moisture,
relative humidity, and temperature. The current data suggest these factors have a similar influence in poultry feed, although they differ in detail. The optimum conditions for production of aflatoxin in broiler feed appeared to be 10 to 13% moisture and 70 to 89% relative humidity which are lower than those reported for stored grains (Christensen, 1975), and 19 to 27 C, which agrees with laboratory findings* of Schroeder and Hein (1967). The greatest difference noted in the present study from prior reports would appear to be in the low moisture level which is lower than the average moisture level in poultry feed manufactured in North Carolina (Tabib et al., 1981), associated here with peak aflatoxin production. The intimate relationship between temperature, moisture, and relative humidity can be used to reconcile the current data. Once the feed in this study entered the house, it started dessicating, as indicated by the correlations between high temperature and low moisture, between long feed age and low moisture, and between high temperature and low relative humidity (Table 8). A correlation between high relative humidity and high aflatoxin concentration rather than between moisture and aflatoxin suggests that the microenvironment supporting aflatoxin production is near the feed surface or it would not be influenced in such a fashion in feed of low moisture content. The early formation of aflatoxin prior to dessication does not seem likely because of the relationship between feed age and aflatoxin concentration (Table 4). Instead, there must be some mechanism that accounts for a moisture optimum of below 13% while the optimal relative humidity is in equilibrium with corn of 16 to 18% moisture (Christensen and Meronuck, 1974). The major difficulty with invoking microenvironments near the feed surface as the site of aflatoxin formation is how to account for dessication in an atmosphere of high relative humidity. This can be done by assuming gradients of water vapor in the house, including the feed, on a permanent or cyclical basis, for example, distillation of water from the feed bin coupled with high relative humidity in the house because of inadequate ventilation. We emphasize that aflatoxin does not occur uniformly throughout feed. Overall, only about 22% of the samples contained detectable aflatoxin and only about 31% of the samples from the poor class of growers were positive. The concentration of aflatoxin in individual
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ochratoxin (Hamilton, Huff, Harris, and Wyatt, unpublished results), while studies such as Stepehnson (1974), which looked only at the farm level, found toxin formation there. These results and conclusions appear to conflict with opinions based on model studies that fungal growth and the production of mycotoxins in stored grains and products is unlikely (Christensen, 1980). The current results have several microbiological implications. The failure of fungal counts to correlate significantly with aflatoxin concentration in feed samples can be explained since only the A. flavus-parasiticus group of the nearly 15,000 species in the Fungi Imperfecti produce aflatoxin (Landecker, 1972), and the medium used to obtain the fungal count was not selective. Thus, it is not likely that measurement of total fungal load would detect shifts within the fungal population. In addition, Tuite (1979) suggested toxic fungi may grow vigorously without producing toxin because the mycotoxin is metabolized, competing fungi use essential nutrients, the environment does not permit toxin formation, and nontoxic strains predominate over toxic strains. The correlation of fungal counts with bacterial counts in a rather dry environment such as feed would appear to be contradictory, since bacterial growth and activity require free water (i.e., free to migrate in capillary spaces as opposed to bound water, which may or may not be available for bacterial growth (Kramer, 1969). The correlation of bacterial counts with coliform counts in feed is also surprising, because coliforms are thought to inhabit aqueous environments. However, another study (Tabib et al., 1981) found increased numbers of coliforms and fungi in feed of suspect microbiological quality. This finding of the present study suggests that the microbial activity under discussion does not occur in the average environment measured but rather that the microorganisms grow in microenvironments not representative of the whole, yet permitting microbial growth commonly associated with the more favorable available water relationships seen in the laboratory. Majumder et al. (1965) showed that moisture migration in sealed containers of grain as a result of temperature gradients encouraged microbial growth in the absence of a net gain in moisture.
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samples ranged from 0 to 196 ppb. The folly of using single samples to assess a possible mycotoxin problem would seem obvious. Trying to duplicate field exposure of animals to aflatoxin in the laboratory would seem to be a formidable task. ACKNOWLEDGMENTS
The technical assistance of D. Joy Osborne and Jim Hutchins is greatly appreciated.
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REFERENCES Barr, A. J., J. H. Goodnight, J. P. Sail, and J. T. Helwig, 1976. A User's Guide to SAS 76. SAS Inst., Inc., Raleigh, NC. Christensen, C. M., 1975. Molds, Mushrooms and Mycotoxins. Univ. Minnesota Press, Minneapolis, MN. Christensen, C. M., 1980. Needed: Research on storage molds in grains, seeds and their products. Plant Dis. 64:1067-1070. .Christensen, C. M., and R. A. Meronuck, 1974. Manual of Fungi in Feeds, Foods, and Cereal Grains. Univ. Minn. Agric. Ext. Serv., St. Paul, MN. Good, R. E., and P. B. Hamilton, 1981. Beneficial effect of reducing the feed residence time in a field problem of suspected moldy feed. Poultry Sci. 60:1403-1405. Hamilton, P. B., 1971. A natural and extremely severe occurrence of aflatoxicosis in laying hens. Poultry Sci. 50:1880-1882. Hamilton, P. B., 1975. Proof of mycotoxicosis being a field problem and a simple method for their control. Poultry Sci. 54:1706-1708. Hamilton, P. B., 1978. Fallacies in our understanding of mycotoxins. J. Food Prot. 41:404—408. Hamilton, P. B., and J. D. Garlich, 1971. Aflatoxin as a possible cause of fatty liver syndrome in laying hens. Poultry Sci. 50:800-804. Hamilton, P. B., and J. R. Harris, 1971. Interaction of aflatoxicosis with Candida albicans infections and other stresses in the chicken. Poultry Sci. 50: 906-912. Hamilton, P. B., H. T. Tung, R. D. Wyatt, and W. E. Donaldson, 1974. Interaction of dietary aflatoxin with some vitamin deficiencies. Poultry Sci. 53:871-877. Horowitz, W., ed., 1980. Methods of Analysis of the Association of Official Analytical Chemists. Assoc. Offic. Anal. Chem., Washington, DC. Huff, W. E., J. A. Doerr, P. B. Hamilton, D. D. Hainason, R. E. Peterson, and A. Ciegler, 1980. Evaluation of bone strength during aflatoxicosis and ochratoxicosis. Appl. Environ. Microbiol. 40: 102-107. Kramer, P. J., 1969. Plant and Soil Water Relationships: A Modern Synthesis. McGraw Hill Book Co., New York, NY. Landecker, E. M., 1972. Fundamentals of the Fungi.
Prentice-Hall, Inc. Englewood Cliffs, NJ. Lillehoj, E. B., and A. Ciegler, 1975. Mycotoxin Synergism. In Microbiology - 1975. D. Schlessinger, ed. Am. Soc. Microbiol., Washington, DC. Majumder, S. K., K. S. Narasimhan, and H.A.B. Parpia, 1965. Microecological factors of microbial spoilage and the occurrence of mycotoxins on stored grains Pages 2 7 - 4 7 in Mycotoxins in Foodstuffs. G. N. Wogan, ed. MIT Press. Amherst, MA. Michael, G. Y., P. Thaxton, and P. B. Hamilton, 1973. Impairment of the reticuloendothelial system of chickens during aflatoxicosis. Poultry Sci. 52: 1206-1207. Pier, A. C , and K. L. Huddleston, 1970. The effect of aflatoxin in immunity in turkeys. I. Impairment of actively acquired resistance to bacterial challenge. Avian Dis. 14:797-809. Schroeder, H. W., 1969. Factors influencing the development of aflatoxins in some field crops. J. Stored Prod. Res. 5:187-192. Schroeder, H. W., and H. Hein, 1967. Aflatoxins: Production of the toxins In vitro in relation to temperature. Appl. Microbiol. 15:441—445. Smith, J. W., and P. B. Hamilton, 1970. Aflatoxicosis in the broiler chicken. Poultry Sci. 49:207-215. Smith, J. W., C. H. Hill, and W. E. Donaldson, 1971. The effect of dietary modifications on aflatoxicosis in the broiler chicken. Poultry Sci. 50:768-774. Smith, R. B., J. H. Griffin, and P. B. Hamilton, 1976. Survey of aflatoxicosis in farm animals. Appl. Environ. Microbiol. 31:385-388. Stephenson, E. L., 1974. The relation of mold to bone disorders in poultry. Proc. North Carolina Poultry Nutr. Conf. 1:1-2. Tabib, Z., F. T. Jones, and P. B. Hamilton, 1981. Microbiological quality of poultry feed and ingredients. Poultry Sci 60:1392-1397. Thaxton, P., H. T. Tung, and P. B. Hamilton, 1974. Immunosuppression in chickens by aflatoxin. Poultry Sci. 53:721-725. Tuite, J., 1979. Field storage conditions for the production of mycotoxins and geographic distributions of some mycotoxins in problems in the United States. In: Interactions of Mycotoxins in Animal Production. Natl. Acad. Sci., Washington, DC. Tung, H. T., and P. B. Hamilton, 1973. Decreased plasma carotenoids during aflatoxicosis. Poultry Sci. 5 2 : 8 0 - 8 3 . Tung, H. T., R. D. Wyatt, P. Thaxton, and P. B. Hamilton. 1973. Impairment of kidney function during aflatoxicosis. Poultry Sci. 52:873-878. Warren, M. F., and P. B. Hamilton, 1980. Intestinal fragility during ochratoxicosis and aflatoxicosis in broiler chickens. Appl. Environ. Microbiol. 40:641-645. Wyatt, R. D., and P. B. Hamilton, 1975. Interaction between aflatoxicosis and a natural infection of chickens with Salmonella. Appl. Microbiol. 30:870-872.