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Interaction of Aflatoxicosis with Candida Albicans Infections and Other Stresses in Chickens12
Interaction of Aflatoxicosis with Candida Albicans Infections and Other Stresses in Chickens1'2 P. B. HAMILTON AND J. R. HARRIS Department of Poultry Science and Department of Microbiology, North Carolina State University, Raleigh, North Carolina 27607 (Received for publication November 16, 1970)
INTRODUCTION
A
Field observations offer another line of evidence that suggests aflatoxin is interacting somehow to the detriment of animal health. Poultry health problems character1 Paper Number 3259 of the Journal Series of the North Carolina State University Agricultural Experiment Station, Raleigh, North Carolina. 2 A preliminary report of this paper was presented at the 59th Annual Meeting of the Poultry Science Association, Knoxville, Tennessee, 1970.
ized by increased mortality and poor growth rates are frequently associated with the presence of aflatoxin in the diet and improve when an aflatoxin-free diet is introduced (Smith and Hamilton, 1970). Yet, the levels of aflatoxin found in surveys of these feed samples frequently are lower than those levels required to produce clinical aflatoxicosis in the laboratory (Smith and Hamilton, 1970; and unpublished results. This suggested that aflatoxin was interacting with other factors. A common stressful factor in poultry husbandry is crop mycosis, a Candida albicans infection, which is characterized by high morbidity, low mortality, decreased growth, and altered mucosal linings of the upper gastrointestinal tract (Yacowitz et al., 1959). Other stresses of practical importance in poultry husbandry are the temperature stresses of heat and cold. It is not uncommon for over 100,000 chickens to die during temperature extremes in North Carolina, and the geographic distribution of these deaths does not correlate necessarily with the recorded temperature and is so unpredictable as to suggest an interacting factor (Harris, unpublished observations.) Our paper reports on the interactions between aflatoxicosis and C. albicans, hot and cold temperature stresses, and the physiological stress of saline drinking water. MATERIALS AND METHODS
Growth of chickens and preparation of diets. Day-old male chicks (Arbor Acre 60 X Peterson) were obtained from a commercial hatchery. They were housed in
906
Downloaded from http://ps.oxfordjournals.org/ at Georgetown University on May 23, 2015
FLATOXICOSIS in chickens has been - characterized mainly by lowered growth rates and inefficient conversion of feed (Carnaghan et al., 1966; Gardiner and Oldroyd, 1965; and Smith and Hamilton, 1970) and the possibility of interaction of aflatoxicosis with other diseases has been studied since the disease was discovered. One of the original reports of aflatoxicosis (Siller and Ostler, 1961) described the isolation of Salmonella from the internal organs of turkeys during field outbreaks of the disease. Brown and Abrams (1965) in a more recent study consistently isolated Salmonella from ducklings and chickens with typical aflatoxicosis. They also noted a hypoproteinemia which included low levels of globulins and suggested that aflatoxicosis made birds more susceptible to Salmonella. Abrams (1965) extended this hypothesis to include other bacterial and viral diseases. When this hypothesis was tested directly, aflatoxicosis and S. gallinarum infections were found to exert their effects on body weight and mortality of chickens independently and without interaction (Smith et al., 1969).
AFLATOXICOSIS AND CANDIDA ALBICANS
907
Downloaded from http://ps.oxfordjournals.org/ at Georgetown University on May 23, 2015
electrically heated batteries where feed and fections in chickens is rated usually on a 0 water were available ad libitum. Four to 4 basis in which the apparent thickness groups of ten birds were fed for each treat- and roughness of the crop lining are the ment for three weeks. Aflatoxicosis was in- subjective criteria (Yacowitz et al., 1959). duced by incorporating known amounts of Because a more objective and quantitative aflatoxin into the commercial starter diet criterion seemed desirable the weight of the from which the usual medications had been crop after excision, washing, and blotting omitted. The diet was analyzed to insure was determined and was expressed relative freedom from extraneous aflatoxin. to the body weight. The validity of using Aflatoxin was produced by growing As- the relative crop weight as a criterion of pergillus flavus NRRL 2999 on rice accord- the severity of C. albicans infections was ing to the method of Shotwell et al. (1966) established by correlating it with the subusing the flasks described by Smith and jective rating of Yacowitz et al. (1959) Hamilton (1969). The moldy rice was and by isolating C. albicans from the crops steamed, dried, and ground to a fine pow- of inoculated birds. The microorganism der. This powder was analyzed for afla- could not be isolated from uninoculated toxin content by extracting with chloro- birds. form and determining its absorbance at 360 Quantitation of temperature and saline nm in a spectrophotometer (Nabney and stresses. The lethal effects of these stresses Nesbitt, 1965) after decolorizing with cop- were quantitated by measuring the mean per carbonate (Wiseman et al., 1967). This survival time of the birds receiving the procedure gave more precise results than stress. The cold temperature stress was a the chromatographic method of Pons et al. slight modification of those used by Sturkie (1966) which was used for identifying and (1946) and Moreng and Shaffner (1951). confirmatory purposes. Weighed amounts The cold stress consisted of immersing of the rice powder were added to the diet the birds momentarily in an ice water bath and at its highest concentration the rice containing a detergent to wet the naturally powder never exceeded one percent of the oily feathers before placing the birds in a diet. forced draft incubator maintained at 4°C. Quantitation of C. albicans infections. and 95% relative humidity. The heat stress The chickens used for the experiments with consisted of placing the birds in a room C. albicans infections were fed a diet com- (Wilson et al., 1966) at 46°C. and 90% posed of one-half glucose and one-half com- relative humidity. The saline stress conmercial starter mash. This diet was nutri- sisted of placing NaCl at one percent contionally inadequate but it permitted the centration in their drinking water. The uniform establishment in the laboratory of chickens exposed to the temperature quantitatively significant C. albicans in- stresses were three weeks old while those fections when otherwise untreated chickens exposed to the saline stress were two weeks were injected with suspensions of C. albi- of age at the beginning. The birds exposed cans. When the chickens were one-week old to these stresses were fed the commercial they were infected by placing in their crops starter diet containing added aflatoxin from 1.0 ml. of 24 hour culture of C. albicans hatching until the end of the experiments. grown at 37°C. in nutrient broth containing one per cent glucose. When the birds RESULTS were three weeks old they were weighed The relationship between aflatoxicosis and killed. The severity of C. albicans in- and C. albicans infections was investigated
908
P. B. HAMILTON AND J. R. HARRIS
£DU
200 _
150
\ ^ \ 5 .
INFECTED
UNINFECTED
^ C r ^ ^
100
1
5.0 AFLAT0XIN(^g/g)
l
10.0
FIG. 1. Effect of afiatoxicosis and Candida albicans infections on the body weight of chickens. The data points are the means of four groups of ten birds receiving that treatment and the vertical bars are the standard error of means.
in a 2 X 6 factorial design experiment for the presence and absence of C. albicans and six concentrations (0, 0.625, 1.25, 2.5, 5.0, and 10.0 (xg./g.) of dietary aflatoxin. The response of the mean body weight is shown in Fig. 1. A factorial analysis of variance of the data revealed that both aflatoxin and C. albicans produced a highly significant (P < 0.01) lowering of the growth rate. There was no overall interaction on body weight between aflatoxin and C. albicans. However, a subdivision of the total interaction into single degrees of freedom portions showed a significant (P < 0.05) 2 X 2 interaction between presence and absence of C. albicans and two levels of aflatoxin (0 and 0.625 jj.g./g.). This interaction at the 0.625 !J.g./g. dose was duplicated in another experiment. The interaction effect, although real and important, did not show up in the overall (2 X 6) interaction test because of dilution effects
i.Or
5.0 AFLATOXIN ( / i g / g )
10.0
FIG. 2. Effect of afiatoxicosis and Candida albicans infections on the relative crop weight of chickens. The data points are the means of four groups of ten birds receiving that treatment and they are the crop weight per 100 grams of body weight. The vertical bars are the standard error of means. These data were obtained in the same experiment depicted in Fig. 1.
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50
with other degrees of freedom where no interaction existed. The response of the crop weight relative to the body weight in the 2 X 6 factorial experiment is shown in Fig. 2. A factorial analysis of variance revealed that the relative crop weight was increased by both aflatoxin and C. albicans in a highly significant (P < 0.01) fashion and that there was a significant (P < 0.0S) positive interaction between them. A partial cause for the interaction between aflatoxin and C. albicans infections is suggested by the data in Table 1. This experiment was a 2 X 2 factorial design for the presence and absence of extra vitamins and 10 pig./g. of aflatoxin in the diet of chickens infected with C. albicans. The data show that the extra vitamins were without significant effect on the body weight or relative crop weight of infected chickens not receiving aflatoxin; however, the extra vitamins did cause a partial but
AFLATOXICOSIS AND CANDIDA ALBICANS
was incorporated into their drinking water is demonstrated in Fig. 5. The mean survival time was decreased about 50 percent by the higher doses and an analysis of variance showed that all doses above 1.25 [Ag./g. decreased highly significantly (P < 0.01) the mean survival time. DISCUSSION These results suggest that many field problems associated with aflatoxin are not true aflatoxicosis but are a syndrome caused by interacting stresses. A good example would be crop mycosis in which C. albicans interacts to produce a damaged crop and a lessened growth rate (Fig. 1 and Fig. 2). A possible explanation for the interaction of C. albicans infections with aflatoxicosis would be that C. albicans is
30 r
TABLE 1.--Interaction of aflatoxin and vitamins
in C albicans infections in chickens Aflatoxin (ppm)
1 + 1 +
0.0 0.0 10.0 10.0
Added Mean body Mean relative vitamins" weightb crop weight0 205 199 92d 1150
0.60 0.63 0.98 d 0.77°
a A standard premixed vitamin combination was obtained from the feed mill that supplied the basal •diet and the vitamins were added at four times the minimum recommended dosage. b Mean body weight in grams at 18 days of age of four groups of ten birds for each treatment. c The relative crop weight is the crop weight per 100 grams of body weight. d This value differs significantly (P <0.01) from the control value in a 2X2 factorial analysis of variance. e A 2X2 factorial analysis of variance showed a significant (P<0.05) interaction between aflatoxin and added vitamins.
< > z
<
5.0 AFLATOXIN ( / i g / g )
10.0
FIG. 3. Effect of aflatoxicosis on the response of chickens to a low temperature stress. Each data point is the mean survival time of four groups of ten birds exposed to 4°C. and 95 percent relative humidity. The vertical bars are the standard error of the mean.
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significant (P < 0.05) reversal of the effects of aflatoxin in the infected chickens. The response to a lethal cold stress by chickens fed graded doses of aflatoxin is shown in Fig. 3. It is obvious that the mean survival time can be lowered to about 50 percent of the control value. An analysis of variance revealed that all doses of aflatoxin above 1.25 u-g./g. produced a significant (P < 0.05) lowering of the mean survival time. Because the variances appeared to be related to the means, another analysis of variance was performed using a logarithmic transformation of the data. The difference patterns obtained were similar to those of the untransformed data. The response to a lethal heat stress of chickens fed graded doses of aflatoxin is shown in Fig. 4. Contrary to the effect with a cold stress, aflatoxicosis makes chickens more resistant to a heat stress. An analysis of variance indicated that all doses of aflatoxin above 1.25 ^.g./g. produced a significant (P < 0.05) increase in the mean survival time. The effect of aflatoxicosis on the survival time of chickens when one percent NaCl
909
910
P. B. HAMILTON AND J. R. HARRIS
I60r
FIG. 4. Effect of aflatoxicosis on the response of chickens to a high temperature stress. Each data point is the mean survival time of four groups of ten chickens exposed to 46°C. and 90 percent relative humidity. The vertical bars are the standard error of the mean.
competing with or making unavailable to the chicken the dietary vitamins and that aflatoxin influences the process in favor of the microorganism. This interpretation is supported by field observations that added dietary vitamins are helpful in some cases of crop mycosis (Harris, unpublished observations). This involvement of aflatoxin with vitamin nutrition should be investigated further. It could be argued that these data on C. albicans infections do not relate to field conditions because the data were obtained with an inadequate diet. However, we could not obtain a reproducible and quantitatively significant infection in the laboratory unless the inadequate diet was used. The idea that nutrition is involved in infections by the opportunistic C. albicans is supported by the previously mentioned field observations that added dietary vitamins are helpful in some cases of crop mycosis. The differential effect of aflatoxicosis on heat and cold stresses was unexpected. The
< > > 3 CO
z
< UJ
5 5.0 AFLATOXIN ( / t g / g )
10.0
FIG. S. Effect of aflatoxicosis on the response of chickens to one percent sodium chloride in their drinking water. Each data point is the mean survival time of four groups of ten chickens receiving that dose of dietary aflatoxin and the vertical bars are the standard error of the mean.
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AFLATOXIN(^g/g)
explanation for these effects probably resides in the observation that young chickens with clinical aflatoxicosis appear to have a reduced amount of depot fat (Smith and Hamilton, 1970). It seems reasonable that fat animals would be more resistant to cold and more susceptible to heat than animals with a below normal amount of depot fat. The physiological stress of saline drinking water is made more severe by aflatoxicosis. It should be noted that during this experiment the chickens drank about ten times the normal volume of water. The deaths were associated probably with a mineral imbalance but it is interesting that only doses above 1.25 [xg./g- produced any effect on the mean survival time. A similar dose threshold was found with growth rate in this experimental system (Smith and Hamilton, 1970) which implies that the physiological defenses of chickens against
A F L A T O X I C O S I S A N D CANDIDA
their environment are generally impaired in clinical aflatoxicosis. The interaction of aflatoxicosis with some common environmental stresses of chickens indicates that aflatoxin possesses profound but insidious properties other than that of the well-documented and feared carcinogenesis (Ciegler and Lillehoj, 1968). This emphasizes anew the importance of mycotoxins to animal and public health.
Some health problems in the poultry industry are associated with the presence of anatoxin in the diet and improve when a diet free of anatoxin is introduced, although the levels of aflatoxin found in surveys of feed samples frequently are lower than those levels required to produce clinical aflatoxicosis in the laboratory. The possibility that aflatoxicosis can interact with other factors was investigated. A 2 X 6 factorial design experiment for the presence and absence of infection with Candida albicans and six levels of dietary aflatoxin showed a significant interaction that resulted in increased crop weight relative to body weight. There was no interaction on body weight except at the lowest level of aflatoxin (0.625 u,g./g.). The addition of extra vitamins to the diet of infected birds caused a partial reversal of the effects of aflatoxin on body weight and relative crop weight. Aflatoxicosis made chickens susceptible to cold and less susceptible to heat as measured by mean survival time at 4°C. and 90 percent relative humidity and 95 percent relative humidity and 46°C. respectively. Aflatoxicosis also decreased the mean survival time of chickens with one percent NaCl in their drinking water. These results suggest that aflatoxicosis can interact with many factors and that many health problems associated with aflatoxin are not true aflatoxicosis but are a syndrome caused by interacting stresses.
911
ACKNOWLED GMENTS
We thank Sharon West and P. D. Justice for technical assistance. REFERENCES Abrams, L., 1965. Mycotoxins in veterinary medicine. South African Med. J. 58: 767-771. Brown, J. M. M., and L. Abrams, 1965. Biochemical studies on alatoxicosis. Onderstepoort J. Vet. Res. 32: 119-146. Carnaghan, R. B. A., G. Lewis, D. S. P. Patterson and R. Allcroft, 1966. Biochemical and pathological aspects of groundnut poisoning in chickens. Pathol. Vet. 3 : 601-615. Ciegler, A., and E. B. Lillehoj, 1968. Mycotoxins. Adv. Appl. Microbiol. 10: 155-219. Gardiner, M. R., and B. Oldroyd, 1965. Avian aflatoxicosis. Australian Vet. J. 4 1 : 272-276. Moreng, R. E., and C. S. Shaffner, 1951. Lethal internal temperatures for the chicken, from fertile eggs to mature bird. Poultry Sci. 30: 255266.
Nabney, J., and B. F. Nesbitt, 1965. A spectrophotometric method of determining the aflatoxins. Analyst, 90: 155-160. Pons, W. A., A. F. Cucullu, L. S. Lee, J. A. Robertson, A. O. Franz and L. A. Goldblatt, 1966. Determination of aflatoxins in agricultural products: Use of aqueous acetone for extraction. J. Assoc. Off. Agr. Chem. 49: 554-562. Shotwell, O. L., C. W. Hesseltine, R. D. Stubblefield and W. G. Sorenson, 1966. Production of aflatoxin on rice. Appl. Microbiol. 14: 425428. Siller, W. G., and D. C. Ostler, 1961. The histopathology of enterohepatic syndrome of turkey poults. Vet. Rec. 73 : 134-138. Smith, J. W., and P. B. Hamilton, 1969. Technique for the aseptic addition of liquid to flask cultures. Appl. Microbiol. 17: 317. Smith, J. W., and P. B. Hamilton, 1970. Aflatoxicosis in the broiler chicken. Poultry Sci. 49: 207-215. Smith, J. W., W. R. Prince and P. B. Hamilton, 1969. Relationship of aflatoxicosis to Salmonella gallinarum infections of chickens. Appl. Microbiol. 18: 946-947. Sturkie, P. D., 1946. Tolerance of adult chickens to hypothermia. Am. J. Physiol. 147: 531-536. Wilson, H. R., A. E. Armas, I. J. Ross, R. W. Dorminey and C. J. Wilcox, 1966. Familial differences in Single Comb White Leghorn chickens in tolerance to high ambient temperature. Poultry Sci. 45: 784-788.
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SUMMARY
ALBICANS
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Wiseman, H. G., W. C. Jacobson and W. C. Harmeyer, 1967. Note on removal of pigments from chloroform extracts of aflatoxin cultures with copper carbonate. J. Assoc. Off. Agri. Chem. SO: 982-983.
Yacowitz, H., S. Wind, W. P. Jambor, N. P. Willett and J. F. Pagano, 1959. Use of mycostatin for the prevention of moniliasis (crop mycosis) in chicks and turkeys. Poultry Sci. 38: 653-660.
Some Physiological Effects of High Environmental Temperatures on the Laying Hen
(Received for publication November 19, 1970)
HOMEOTHERMY conductance of the tissues, the body surIRDS are homeotherms and so are less face area and the degree of subcutaneous vulnerable to environmental temper- vasodilation. The second stage involves both ature changes than poikilotherms in respect the transfer of sensible heat from the skin of both functional efficiency and danger of surface, through the feathers and boundary tissue damage. The price that has to be layer of still air to the outside environment paid for the benefits conferred by homeo- by conduction, convection or radiation or thermy is that body temperature cannot be the loss of insensible heat, by evaporation allowed to fluctuate beyond relatively nar- from the skin and the lungs. row limits without deterioration in normal The mechanism of thermoregulation in functional efficiency. Within a limited, if ill poultry has not been fully elucidated. In defined, environmental temperature range, humans the thermoregulatory centre of the the laying hen is able to balance thermo- body, which is located in the hypothalagenesis and thermolysis so that its body mus, is comprised of two anatomically distemperature remains at the optimum for tinct subcentres, one of which is responsinormal body functions. ble for heat conservation (e.g. by cutaneComplete uniformity of body tempera- ous vaso-constriction and shivering) while ture is possible only if no heat exchange oc- the other is concerned with heat dissipation curs between the body and its environment. (e.g. by vaso-dilation and sweating) (LeitBirds, however, constantly produce heat head and Lind, 1964). Leithhead and Lind and lose it to the environment so that there suggest that the hypothalamus responds diis a thermal gradient from the warm inte- rectly to local brain temperature changes rior (core) to the cooler surface (shell). induced by variations in the temperature of Sensible heat constantly flows from the the blood supplying the brain, as well as to deep body centre to the outside in two indirect afferent impulses from thermorestages, from the core to the surface of the ceptors. This suggestion was confirmed by body and from the skin surface to the am- Bligh (1966) who reviewed the literature bient air. The rate of flow in the first stage dealing with thermoregulation in mammals. is dependent upon the temperature gradient He concluded that the thermosensitivity of between the deep centre and the skin, the well defined areas, within the hypothalmic
B
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A. J. SMITH AND J. OLIVER Department of Agriculture, University of Rhodesia, P.O. Box MP. 167, Mount Pleasant, Salisbury, Rhodesia
INTRODUCTION
A
Field observations offer another line of evidence that suggests aflatoxin is interacting somehow to the detriment of animal health. Poultry health problems character1 Paper Number 3259 of the Journal Series of the North Carolina State University Agricultural Experiment Station, Raleigh, North Carolina. 2 A preliminary report of this paper was presented at the 59th Annual Meeting of the Poultry Science Association, Knoxville, Tennessee, 1970.
ized by increased mortality and poor growth rates are frequently associated with the presence of aflatoxin in the diet and improve when an aflatoxin-free diet is introduced (Smith and Hamilton, 1970). Yet, the levels of aflatoxin found in surveys of these feed samples frequently are lower than those levels required to produce clinical aflatoxicosis in the laboratory (Smith and Hamilton, 1970; and unpublished results. This suggested that aflatoxin was interacting with other factors. A common stressful factor in poultry husbandry is crop mycosis, a Candida albicans infection, which is characterized by high morbidity, low mortality, decreased growth, and altered mucosal linings of the upper gastrointestinal tract (Yacowitz et al., 1959). Other stresses of practical importance in poultry husbandry are the temperature stresses of heat and cold. It is not uncommon for over 100,000 chickens to die during temperature extremes in North Carolina, and the geographic distribution of these deaths does not correlate necessarily with the recorded temperature and is so unpredictable as to suggest an interacting factor (Harris, unpublished observations.) Our paper reports on the interactions between aflatoxicosis and C. albicans, hot and cold temperature stresses, and the physiological stress of saline drinking water. MATERIALS AND METHODS
Growth of chickens and preparation of diets. Day-old male chicks (Arbor Acre 60 X Peterson) were obtained from a commercial hatchery. They were housed in
906
Downloaded from http://ps.oxfordjournals.org/ at Georgetown University on May 23, 2015
FLATOXICOSIS in chickens has been - characterized mainly by lowered growth rates and inefficient conversion of feed (Carnaghan et al., 1966; Gardiner and Oldroyd, 1965; and Smith and Hamilton, 1970) and the possibility of interaction of aflatoxicosis with other diseases has been studied since the disease was discovered. One of the original reports of aflatoxicosis (Siller and Ostler, 1961) described the isolation of Salmonella from the internal organs of turkeys during field outbreaks of the disease. Brown and Abrams (1965) in a more recent study consistently isolated Salmonella from ducklings and chickens with typical aflatoxicosis. They also noted a hypoproteinemia which included low levels of globulins and suggested that aflatoxicosis made birds more susceptible to Salmonella. Abrams (1965) extended this hypothesis to include other bacterial and viral diseases. When this hypothesis was tested directly, aflatoxicosis and S. gallinarum infections were found to exert their effects on body weight and mortality of chickens independently and without interaction (Smith et al., 1969).
AFLATOXICOSIS AND CANDIDA ALBICANS
907
Downloaded from http://ps.oxfordjournals.org/ at Georgetown University on May 23, 2015
electrically heated batteries where feed and fections in chickens is rated usually on a 0 water were available ad libitum. Four to 4 basis in which the apparent thickness groups of ten birds were fed for each treat- and roughness of the crop lining are the ment for three weeks. Aflatoxicosis was in- subjective criteria (Yacowitz et al., 1959). duced by incorporating known amounts of Because a more objective and quantitative aflatoxin into the commercial starter diet criterion seemed desirable the weight of the from which the usual medications had been crop after excision, washing, and blotting omitted. The diet was analyzed to insure was determined and was expressed relative freedom from extraneous aflatoxin. to the body weight. The validity of using Aflatoxin was produced by growing As- the relative crop weight as a criterion of pergillus flavus NRRL 2999 on rice accord- the severity of C. albicans infections was ing to the method of Shotwell et al. (1966) established by correlating it with the subusing the flasks described by Smith and jective rating of Yacowitz et al. (1959) Hamilton (1969). The moldy rice was and by isolating C. albicans from the crops steamed, dried, and ground to a fine pow- of inoculated birds. The microorganism der. This powder was analyzed for afla- could not be isolated from uninoculated toxin content by extracting with chloro- birds. form and determining its absorbance at 360 Quantitation of temperature and saline nm in a spectrophotometer (Nabney and stresses. The lethal effects of these stresses Nesbitt, 1965) after decolorizing with cop- were quantitated by measuring the mean per carbonate (Wiseman et al., 1967). This survival time of the birds receiving the procedure gave more precise results than stress. The cold temperature stress was a the chromatographic method of Pons et al. slight modification of those used by Sturkie (1966) which was used for identifying and (1946) and Moreng and Shaffner (1951). confirmatory purposes. Weighed amounts The cold stress consisted of immersing of the rice powder were added to the diet the birds momentarily in an ice water bath and at its highest concentration the rice containing a detergent to wet the naturally powder never exceeded one percent of the oily feathers before placing the birds in a diet. forced draft incubator maintained at 4°C. Quantitation of C. albicans infections. and 95% relative humidity. The heat stress The chickens used for the experiments with consisted of placing the birds in a room C. albicans infections were fed a diet com- (Wilson et al., 1966) at 46°C. and 90% posed of one-half glucose and one-half com- relative humidity. The saline stress conmercial starter mash. This diet was nutri- sisted of placing NaCl at one percent contionally inadequate but it permitted the centration in their drinking water. The uniform establishment in the laboratory of chickens exposed to the temperature quantitatively significant C. albicans in- stresses were three weeks old while those fections when otherwise untreated chickens exposed to the saline stress were two weeks were injected with suspensions of C. albi- of age at the beginning. The birds exposed cans. When the chickens were one-week old to these stresses were fed the commercial they were infected by placing in their crops starter diet containing added aflatoxin from 1.0 ml. of 24 hour culture of C. albicans hatching until the end of the experiments. grown at 37°C. in nutrient broth containing one per cent glucose. When the birds RESULTS were three weeks old they were weighed The relationship between aflatoxicosis and killed. The severity of C. albicans in- and C. albicans infections was investigated
908
P. B. HAMILTON AND J. R. HARRIS
£DU
200 _
150
\ ^ \ 5 .
INFECTED
UNINFECTED
^ C r ^ ^
100
1
5.0 AFLAT0XIN(^g/g)
l
10.0
FIG. 1. Effect of afiatoxicosis and Candida albicans infections on the body weight of chickens. The data points are the means of four groups of ten birds receiving that treatment and the vertical bars are the standard error of means.
in a 2 X 6 factorial design experiment for the presence and absence of C. albicans and six concentrations (0, 0.625, 1.25, 2.5, 5.0, and 10.0 (xg./g.) of dietary aflatoxin. The response of the mean body weight is shown in Fig. 1. A factorial analysis of variance of the data revealed that both aflatoxin and C. albicans produced a highly significant (P < 0.01) lowering of the growth rate. There was no overall interaction on body weight between aflatoxin and C. albicans. However, a subdivision of the total interaction into single degrees of freedom portions showed a significant (P < 0.05) 2 X 2 interaction between presence and absence of C. albicans and two levels of aflatoxin (0 and 0.625 jj.g./g.). This interaction at the 0.625 !J.g./g. dose was duplicated in another experiment. The interaction effect, although real and important, did not show up in the overall (2 X 6) interaction test because of dilution effects
i.Or
5.0 AFLATOXIN ( / i g / g )
10.0
FIG. 2. Effect of afiatoxicosis and Candida albicans infections on the relative crop weight of chickens. The data points are the means of four groups of ten birds receiving that treatment and they are the crop weight per 100 grams of body weight. The vertical bars are the standard error of means. These data were obtained in the same experiment depicted in Fig. 1.
Downloaded from http://ps.oxfordjournals.org/ at Georgetown University on May 23, 2015
50
with other degrees of freedom where no interaction existed. The response of the crop weight relative to the body weight in the 2 X 6 factorial experiment is shown in Fig. 2. A factorial analysis of variance revealed that the relative crop weight was increased by both aflatoxin and C. albicans in a highly significant (P < 0.01) fashion and that there was a significant (P < 0.0S) positive interaction between them. A partial cause for the interaction between aflatoxin and C. albicans infections is suggested by the data in Table 1. This experiment was a 2 X 2 factorial design for the presence and absence of extra vitamins and 10 pig./g. of aflatoxin in the diet of chickens infected with C. albicans. The data show that the extra vitamins were without significant effect on the body weight or relative crop weight of infected chickens not receiving aflatoxin; however, the extra vitamins did cause a partial but
AFLATOXICOSIS AND CANDIDA ALBICANS
was incorporated into their drinking water is demonstrated in Fig. 5. The mean survival time was decreased about 50 percent by the higher doses and an analysis of variance showed that all doses above 1.25 [Ag./g. decreased highly significantly (P < 0.01) the mean survival time. DISCUSSION These results suggest that many field problems associated with aflatoxin are not true aflatoxicosis but are a syndrome caused by interacting stresses. A good example would be crop mycosis in which C. albicans interacts to produce a damaged crop and a lessened growth rate (Fig. 1 and Fig. 2). A possible explanation for the interaction of C. albicans infections with aflatoxicosis would be that C. albicans is
30 r
TABLE 1.--Interaction of aflatoxin and vitamins
in C albicans infections in chickens Aflatoxin (ppm)
1 + 1 +
0.0 0.0 10.0 10.0
Added Mean body Mean relative vitamins" weightb crop weight0 205 199 92d 1150
0.60 0.63 0.98 d 0.77°
a A standard premixed vitamin combination was obtained from the feed mill that supplied the basal •diet and the vitamins were added at four times the minimum recommended dosage. b Mean body weight in grams at 18 days of age of four groups of ten birds for each treatment. c The relative crop weight is the crop weight per 100 grams of body weight. d This value differs significantly (P <0.01) from the control value in a 2X2 factorial analysis of variance. e A 2X2 factorial analysis of variance showed a significant (P<0.05) interaction between aflatoxin and added vitamins.
< > z
<
5.0 AFLATOXIN ( / i g / g )
10.0
FIG. 3. Effect of aflatoxicosis on the response of chickens to a low temperature stress. Each data point is the mean survival time of four groups of ten birds exposed to 4°C. and 95 percent relative humidity. The vertical bars are the standard error of the mean.
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significant (P < 0.05) reversal of the effects of aflatoxin in the infected chickens. The response to a lethal cold stress by chickens fed graded doses of aflatoxin is shown in Fig. 3. It is obvious that the mean survival time can be lowered to about 50 percent of the control value. An analysis of variance revealed that all doses of aflatoxin above 1.25 u-g./g. produced a significant (P < 0.05) lowering of the mean survival time. Because the variances appeared to be related to the means, another analysis of variance was performed using a logarithmic transformation of the data. The difference patterns obtained were similar to those of the untransformed data. The response to a lethal heat stress of chickens fed graded doses of aflatoxin is shown in Fig. 4. Contrary to the effect with a cold stress, aflatoxicosis makes chickens more resistant to a heat stress. An analysis of variance indicated that all doses of aflatoxin above 1.25 ^.g./g. produced a significant (P < 0.05) increase in the mean survival time. The effect of aflatoxicosis on the survival time of chickens when one percent NaCl
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FIG. 4. Effect of aflatoxicosis on the response of chickens to a high temperature stress. Each data point is the mean survival time of four groups of ten chickens exposed to 46°C. and 90 percent relative humidity. The vertical bars are the standard error of the mean.
competing with or making unavailable to the chicken the dietary vitamins and that aflatoxin influences the process in favor of the microorganism. This interpretation is supported by field observations that added dietary vitamins are helpful in some cases of crop mycosis (Harris, unpublished observations). This involvement of aflatoxin with vitamin nutrition should be investigated further. It could be argued that these data on C. albicans infections do not relate to field conditions because the data were obtained with an inadequate diet. However, we could not obtain a reproducible and quantitatively significant infection in the laboratory unless the inadequate diet was used. The idea that nutrition is involved in infections by the opportunistic C. albicans is supported by the previously mentioned field observations that added dietary vitamins are helpful in some cases of crop mycosis. The differential effect of aflatoxicosis on heat and cold stresses was unexpected. The
< > > 3 CO
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< UJ
5 5.0 AFLATOXIN ( / t g / g )
10.0
FIG. S. Effect of aflatoxicosis on the response of chickens to one percent sodium chloride in their drinking water. Each data point is the mean survival time of four groups of ten chickens receiving that dose of dietary aflatoxin and the vertical bars are the standard error of the mean.
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AFLATOXIN(^g/g)
explanation for these effects probably resides in the observation that young chickens with clinical aflatoxicosis appear to have a reduced amount of depot fat (Smith and Hamilton, 1970). It seems reasonable that fat animals would be more resistant to cold and more susceptible to heat than animals with a below normal amount of depot fat. The physiological stress of saline drinking water is made more severe by aflatoxicosis. It should be noted that during this experiment the chickens drank about ten times the normal volume of water. The deaths were associated probably with a mineral imbalance but it is interesting that only doses above 1.25 [xg./g- produced any effect on the mean survival time. A similar dose threshold was found with growth rate in this experimental system (Smith and Hamilton, 1970) which implies that the physiological defenses of chickens against
A F L A T O X I C O S I S A N D CANDIDA
their environment are generally impaired in clinical aflatoxicosis. The interaction of aflatoxicosis with some common environmental stresses of chickens indicates that aflatoxin possesses profound but insidious properties other than that of the well-documented and feared carcinogenesis (Ciegler and Lillehoj, 1968). This emphasizes anew the importance of mycotoxins to animal and public health.
Some health problems in the poultry industry are associated with the presence of anatoxin in the diet and improve when a diet free of anatoxin is introduced, although the levels of aflatoxin found in surveys of feed samples frequently are lower than those levels required to produce clinical aflatoxicosis in the laboratory. The possibility that aflatoxicosis can interact with other factors was investigated. A 2 X 6 factorial design experiment for the presence and absence of infection with Candida albicans and six levels of dietary aflatoxin showed a significant interaction that resulted in increased crop weight relative to body weight. There was no interaction on body weight except at the lowest level of aflatoxin (0.625 u,g./g.). The addition of extra vitamins to the diet of infected birds caused a partial reversal of the effects of aflatoxin on body weight and relative crop weight. Aflatoxicosis made chickens susceptible to cold and less susceptible to heat as measured by mean survival time at 4°C. and 90 percent relative humidity and 95 percent relative humidity and 46°C. respectively. Aflatoxicosis also decreased the mean survival time of chickens with one percent NaCl in their drinking water. These results suggest that aflatoxicosis can interact with many factors and that many health problems associated with aflatoxin are not true aflatoxicosis but are a syndrome caused by interacting stresses.
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ACKNOWLED GMENTS
We thank Sharon West and P. D. Justice for technical assistance. REFERENCES Abrams, L., 1965. Mycotoxins in veterinary medicine. South African Med. J. 58: 767-771. Brown, J. M. M., and L. Abrams, 1965. Biochemical studies on alatoxicosis. Onderstepoort J. Vet. Res. 32: 119-146. Carnaghan, R. B. A., G. Lewis, D. S. P. Patterson and R. Allcroft, 1966. Biochemical and pathological aspects of groundnut poisoning in chickens. Pathol. Vet. 3 : 601-615. Ciegler, A., and E. B. Lillehoj, 1968. Mycotoxins. Adv. Appl. Microbiol. 10: 155-219. Gardiner, M. R., and B. Oldroyd, 1965. Avian aflatoxicosis. Australian Vet. J. 4 1 : 272-276. Moreng, R. E., and C. S. Shaffner, 1951. Lethal internal temperatures for the chicken, from fertile eggs to mature bird. Poultry Sci. 30: 255266.
Nabney, J., and B. F. Nesbitt, 1965. A spectrophotometric method of determining the aflatoxins. Analyst, 90: 155-160. Pons, W. A., A. F. Cucullu, L. S. Lee, J. A. Robertson, A. O. Franz and L. A. Goldblatt, 1966. Determination of aflatoxins in agricultural products: Use of aqueous acetone for extraction. J. Assoc. Off. Agr. Chem. 49: 554-562. Shotwell, O. L., C. W. Hesseltine, R. D. Stubblefield and W. G. Sorenson, 1966. Production of aflatoxin on rice. Appl. Microbiol. 14: 425428. Siller, W. G., and D. C. Ostler, 1961. The histopathology of enterohepatic syndrome of turkey poults. Vet. Rec. 73 : 134-138. Smith, J. W., and P. B. Hamilton, 1969. Technique for the aseptic addition of liquid to flask cultures. Appl. Microbiol. 17: 317. Smith, J. W., and P. B. Hamilton, 1970. Aflatoxicosis in the broiler chicken. Poultry Sci. 49: 207-215. Smith, J. W., W. R. Prince and P. B. Hamilton, 1969. Relationship of aflatoxicosis to Salmonella gallinarum infections of chickens. Appl. Microbiol. 18: 946-947. Sturkie, P. D., 1946. Tolerance of adult chickens to hypothermia. Am. J. Physiol. 147: 531-536. Wilson, H. R., A. E. Armas, I. J. Ross, R. W. Dorminey and C. J. Wilcox, 1966. Familial differences in Single Comb White Leghorn chickens in tolerance to high ambient temperature. Poultry Sci. 45: 784-788.
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SUMMARY
ALBICANS
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Wiseman, H. G., W. C. Jacobson and W. C. Harmeyer, 1967. Note on removal of pigments from chloroform extracts of aflatoxin cultures with copper carbonate. J. Assoc. Off. Agri. Chem. SO: 982-983.
Yacowitz, H., S. Wind, W. P. Jambor, N. P. Willett and J. F. Pagano, 1959. Use of mycostatin for the prevention of moniliasis (crop mycosis) in chicks and turkeys. Poultry Sci. 38: 653-660.
Some Physiological Effects of High Environmental Temperatures on the Laying Hen
(Received for publication November 19, 1970)
HOMEOTHERMY conductance of the tissues, the body surIRDS are homeotherms and so are less face area and the degree of subcutaneous vulnerable to environmental temper- vasodilation. The second stage involves both ature changes than poikilotherms in respect the transfer of sensible heat from the skin of both functional efficiency and danger of surface, through the feathers and boundary tissue damage. The price that has to be layer of still air to the outside environment paid for the benefits conferred by homeo- by conduction, convection or radiation or thermy is that body temperature cannot be the loss of insensible heat, by evaporation allowed to fluctuate beyond relatively nar- from the skin and the lungs. row limits without deterioration in normal The mechanism of thermoregulation in functional efficiency. Within a limited, if ill poultry has not been fully elucidated. In defined, environmental temperature range, humans the thermoregulatory centre of the the laying hen is able to balance thermo- body, which is located in the hypothalagenesis and thermolysis so that its body mus, is comprised of two anatomically distemperature remains at the optimum for tinct subcentres, one of which is responsinormal body functions. ble for heat conservation (e.g. by cutaneComplete uniformity of body tempera- ous vaso-constriction and shivering) while ture is possible only if no heat exchange oc- the other is concerned with heat dissipation curs between the body and its environment. (e.g. by vaso-dilation and sweating) (LeitBirds, however, constantly produce heat head and Lind, 1964). Leithhead and Lind and lose it to the environment so that there suggest that the hypothalamus responds diis a thermal gradient from the warm inte- rectly to local brain temperature changes rior (core) to the cooler surface (shell). induced by variations in the temperature of Sensible heat constantly flows from the the blood supplying the brain, as well as to deep body centre to the outside in two indirect afferent impulses from thermorestages, from the core to the surface of the ceptors. This suggestion was confirmed by body and from the skin surface to the am- Bligh (1966) who reviewed the literature bient air. The rate of flow in the first stage dealing with thermoregulation in mammals. is dependent upon the temperature gradient He concluded that the thermosensitivity of between the deep centre and the skin, the well defined areas, within the hypothalmic
B
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A. J. SMITH AND J. OLIVER Department of Agriculture, University of Rhodesia, P.O. Box MP. 167, Mount Pleasant, Salisbury, Rhodesia