Q U A N T I T A T I O N OF ENDOTOXIN IN CELL-FREE RUMEN FLUID OF CATTLE 1 T. G. Nagaraja, E. E. Bartley, L. R. Fina, II. D. Anthony, S. M. Dennis and R. M. Bechtle
Kansas State University, Manhattan 6 6 5 0 6
SUMMARY
Free endotoxin in rumen fluid of cattle was demonstrated by death of mice injected intrapcritoneally with cell-free rumen fluid fraction potentiated with 20 ~tg actinomycin D (Am D). The toxic principle was resistant to heat, was inactivated by acid and alkali, was protected by cortisone and was not lethal to endotoxintolerant mice. Adapting the procedure to quantitate free endotoxin concentration in rumen fluid in cattle, we found the concentration of free endotoxin to be considerably higher in grain-fed than in hay-fed cattle. Concentration was not related to the number of gram-negative bacteria in the tureen; hcnce we postulate that certain factors, as yet unknown, may favor release of frec endotoxin from intact gramnegative bacteria. (Key Words: Endotoxin, Rumen Fluid, Cattle.)
stances extracted from cell-free rumen fluid of cattle possess some of the classical properties of endotoxin found in such gram-negative bacteria as Eschericbia coil (Mullenax et al., 1966; Nagaraja et al., 1976). Having demonstrated that cell-free rumen fluid exhibits certain biological characteristics similar to endotoxins of gram-negative bacteria, we (Nagaraja et al., 1977) observed a quantitative difference in concentration of endotoxin in rumen fluids of cattle fed hay or grain. This report deals with a method to assay free endotoxin in rumen fluid of cattle based on the lethal effect on mice of cell-free rumen fluid potentiated with actinomycin D. Also, after obtaining evidence to confirm that the lethal agent was endotoxin we determined endotoxin concentrations in rumen fluid of cattle fed hay, grain or pasture. MATERIALS AND METHODS
INTRODUCTION
Effect o f lleat on Lethality o f Cell-free R u m e n Iquid. A rumen-fistulated adult Jersey cow was used as a source of rumen fluid. The cow was fed daily 5.44 kg of a 14% crude protein grain ration containing a mixture of sorghum grain (IRN 4-04-378), corn (IRN 4-02-931), soybean meal (IRN 5-04-004), Starea 2, dicalcium phosphate (1RN 6-01-080), salt (IRN 6-04-152), and a vitamin A and D supplement. Rumen fluid was obtained between 3 and 6 hr post feeding and immediately strained through four layers of cheesecloth. The strained tureen fluid was centrifuged at 450 g for 5 minutes. The supernatant containing bacteria was centrifuged at 25,000 g for 25 minutes. The resulting supernatant was determined cell-free by microscopic examination ~Contribution No. 78-24j, Department of Animal Sciences and Industry, Division of Biology (Micro- before being assayed. biology Section) and Veterinary Diagnostic LaboraRandom bred albino mice (20 to 25 g) of tory, Kansas Agricultural Experiment Station, Manhatboth sexes were fed a commercial stock diet tan. 2Starea is an extrusion processed mixture of grain (Purina Laboratory Chow, Ralston Purina Co., St. Louis, MO). Feed and water were provided and urea (registered trademark 860255, U.S. Patent No. 364.2489). ad libitum. Serial twofold dilutions of cell-free 1759 Rumen flora contains many gram-negative bacteria, a suspected source of endotoxin. Endotoxins may play an important role in the pathogenesis of certain diet-induced diseases like lactic acidosis, bloat and the sudden death syndrome (Dougherty and Cello, 1949, 1953; Mullenax et al., 1966; Dougherty et al., 1975; Dougherty, 1976; l[uber, 1976; Nagaraja et al., 1978). To produce disease, rumen bacterial endotoxins must be released from the bacteria and absorbed from the gastro-intestinal tract into the system. Therefore, free endotoxins should be detectable in cell-free rumen fluid. Sub-
JOURNAL OF ANIMAL SCIENCE, Vol. 46, No. 6, 1978 Downloaded from https://academic.oup.com/jas/article-abstract/46/6/1759/4699315 by guest on 09 April 2018
1760
NAGARAJA ET AL.
rumen fluid (minimum of four dilutions) were made in pyrogen-free saline and injected into groups of five mice each. This process was repeated a minimum of three times. The size of inoculum per mouse was .5 ml mixed with 20 gig of actinomycin D (Am D C o s m o g e n | Lots 1203T or 0724V. Merck Sharp and Dohme, West Point, PA). All injections were given intraperitoneally with sterile disposable syringes. Deaths were recorded up to 2 days. The LDs 0 was calculated by the Probit procedure with a modified Gauss-Newton logarithm to estimate the maximum likelihood of estimates of the intercept, slope and threshold response rate of the biological assay data (Finney, 11971). Cell-free rumen fluid was heated in a boiling water bath maintained at 100 C for 30, 180 or 360 minutes. Also, another sample was autoclaved (121 C, 15 psi for 20 min). The LDs 0 of the heated cell-free rumen fluid potentiated with Am D was estimated, and the change in LDs0 from the control was calculated.
F4"fect o f Acid Hydrolysis on Lethality o f Cell-]tee Rumen Fluid. One hundred ml of cell-free rumen fluid was treated with an equal volume of .2 N acetic acid. The mixture was kept in a boiling water bath for 30, 60, or 120 minutes. When removed from the bath, the mixture was quickly cooled to room temperature and neutralized with 10 N NaOH to the initial pH of rumen fluid. A control was prepared by adding acetic acid to cell-free rumen fluid and then neutralizing (without boiling) with 10 N NaOH to original pH. The neutralized heated or unheated cell-free rumen fluids were then concentrated in vacuum to obtain the original volume for the LDs 0 assay in mice.
Effect o f Alkaline tlydrolysis on Letbality of Cell-free Rumen Fluid. Cell-free rumen fluid was treated with .1 N NaOH and incubated at room temperature. At 3 or 6 or 24 hr, the NaOH-treated rumen fluid was neutralized with acetic acid to the initial pH and concentrated in vacuum to obtain the original volume. The control sample was prepared similarly by neutralizing NaOH-treated cell-free rumen fluid with acetic acid at 0 hours. The LDs0 was estimated in mice, and change in LDs0 from the control was calculated.
Protective Effect o f Cortisone on Lethality o f Cell-free Rumen Fluid. Groups of mice were injected subcutaneously with 5 mg (per mouse)
of cortisone (Cortone| acetate Merck, Sharp and Dohme, West Point, PA). The mouse LDs0 of cell-free rumen fluid was determined at 6 or 1 hr before, at the same time, and at 1 or 6 hr after injecting cortisone acetate. A control group received no cortisone acetate.
Lethal Effect o f Cell-free Rumen Fluid on Mice Made Tolerant to E. coil Endotoxin. The test was according to the procedure of Albizo and Surgallo (1970). One microgram of E. coli endotoxin (0111:B4 Difco Laboratories, Detroit, MI) contained in .1 ml of sterile pyrogenfree saline was injected intraperitoneally into each mouse (weight 20 to 25 g) on day one of the experiment. After 72 hr each mouse was injected with 10 gig of the same endotoxin. Also, each mouse in the control group (containing the same number of mice) was injected intraperitoneally with .1 ml of pyrogen-free saline on day one and .5 ml in 72 hours. On the 5th day, groups of control and test mice were injected with E. coil or Salmonella typhosa (0901 Difco) endotoxin with or without Am D or cell-free rumen fluid (with Am D) from cattle fed hay or grain. The dosages used are shown in table 5. Deaths were recorded up to 48 hours.
Concentration o f Free Endotoxin in Rumen Fluid from Cattle Fed All Hay or All Grain or ltigh-concentrate Ration. Two rumen-fistulated adult Jersey cows were used. One was fed daily 5.44 kg alfalfa hay (1RN 1-00-063) and the other received daily 5.44 kg of a 14% crude protein grain ration similar to that used previously. Rumen fluid samples were obtained at 0, 3, 6, 12 and 24 hr post feeding. The LDs 0 of cell-free rumen fluid potentiated with 20 gig Am D was determined in mice. The concentration of endotoxin in rumen fluid expressed as rumen bacterial endotoxin activity was calculated on the basis that LDs 0 of cell-free rumen fluid should contain .86 gig of endotoxin which is the established LDs0 of rumen bacterial endotoxin (Nagaraja et al., 1978). Total bacterial numbers in rumen fluid were determined by diluting samples with 10% formal saline and counting in a Petroff-Hausser bacterial counting cl~amber (C. A. Hausser and Son, Philadelphia, PA). Percentages of gram-negative and grampositive bacteria were estimated by counting a minimum of 1,000 cells fr~)m a gram-stained smear. The smear was made by spreading evenly 5 gil of diluted rumen fluid on an area of 1 cm 2 (Warner, 1962). All counts were done in duplicate and were averaged.
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ENDOTOXIN IN CATTLE RUMEN FLUID Also, rumen fluid samples were obtained 6 hr post feeding from five rumen-fistulated cows and five heifers adapted to an all roughage ration of alfalfa hay; from five steers (400 to 450 kg) fed a high-concentrate low roughage ration for more than 100 days; and from five steers (375 to 400 kg) fed an all concentrate ration for more than 100 days. The steers on the high-concentrate low-roughage ration were fed twice daily ad libitum quantities (as-fed basis) a ration of 24.0% sorghum silage (31.8% dry matter), 70.2% rolled sorghum grain, 4.31% soybean meal, .12% urea, (IRN 5-05-070),. 37% limestone (IRN 6-02-632), . 3% dicalcium phosphate, .5% salt, .05% trace mineral (Z-5 Calcium Carbonate Co., Quincy, IL), .04% aureomycin (22.2 g/kg), .01% vitamin A (30,000 IU/g). The steers fed an all concentrate ration were fed 8.33 kg (divided into two feedings) of the following: 62% rolled sorghum grain, 25% cracked wheat (IRN 4-05-268), 10% Starea, 2% dicalcium phosphate, .5% trace mineralized salt, and .5% vitamin A and D supplement (1,000,000 units of A and D per kilogram supplement). The LDs0 of the cell-free rumen fluid, endotoxin activity, total bacterial number, percentages of gram-negative and positive bacteria were determined as before. RESULTS AND DISCUSSION
Effect of Heat on Lethality o f Cell-free Rumen Fluid. Unheated cell-free rumen fluid was lethal to mice (LDs 0 = .054 ml, table 1). Heating cell-free rumen fluid at 100 C for 30 rain did n o t change the LDs0. However, pro-
1761
longed heating (3 or 6 hr) reduced the toxicity of cell-free rumen fluid by a factor of 1.7 and 3.0, respectively. Autoclaving cell-free rumen fluid caused a small decrease in toxicity. Therefore, the toxic principle responsible for the death of mice apparently is resistant to heat (at least for 30 min). Extreme heat tolerance was one of the earliest methods used to distinguish endotoxins from other bacterial toxins, but no uniformity has been noted in the m i n i m u m temperature needed to destroy endotoxin (Milner et al., 1971). Neter et al. (1956) observed that heating Salmonella abortus equi endotoxin at 100 C for 2.5 hr did not alter its toxicity.
Effect o f Acid Hydrolysis on Lethality of Cell-free Rumen Fluid. The toxicity of cell-free rumen fluid boiled with .2 N acetic acid decreased progressively with time (table 2). The LDs0 increased about two and a half times after hydrolyzing for 30 min and almost four times after 2 hours. Mild acid apparently inactivates the toxic principle in rumen fluid. The loss of biological activity (including mouse lethality) of endotoxin when exposed to acid at elevated temperature has been shown (Sultzer, 1971). Tal and Goebel (1950) showed that Shigella endotoxin treated with 1% acetic acid at 100 C reduced mouse lethality by four times within 60 minutes. Haskins et al. (1961), who studied in detail the effects of acid hydrolysis on biological activities of Salmonella enteritidis endotoxin, observed that within 15 min the lethality of the endotoxin in mice decreased dramatically. In our study, acid hydrolysis of the cell-free rumen fluid for 2 hr did not render it completely nontoxic.
TABLE 1. EFFECT OF HEAT ON LETHALITY OF CELL-FREE RUMEN FLUID PLUS ACTINOMYCIND ON MICEa
Heat treatment
Duration of treatment
None (control) 100 c 100 C 100 C Autoclaved
(min) 0 30 180 360 20
LDs o
Increase in LD 5o
(ml) .054 +- .004b .053 +- .011
...
.093 + .034*
117x
.164 + .028* .089 -+.018"
3.0x 1.6x
aAverage of three experiments. bMean + standard error.
*Significantly different from control (P
Kansas State University, Manhattan 6 6 5 0 6
SUMMARY
Free endotoxin in rumen fluid of cattle was demonstrated by death of mice injected intrapcritoneally with cell-free rumen fluid fraction potentiated with 20 ~tg actinomycin D (Am D). The toxic principle was resistant to heat, was inactivated by acid and alkali, was protected by cortisone and was not lethal to endotoxintolerant mice. Adapting the procedure to quantitate free endotoxin concentration in rumen fluid in cattle, we found the concentration of free endotoxin to be considerably higher in grain-fed than in hay-fed cattle. Concentration was not related to the number of gram-negative bacteria in the tureen; hcnce we postulate that certain factors, as yet unknown, may favor release of frec endotoxin from intact gramnegative bacteria. (Key Words: Endotoxin, Rumen Fluid, Cattle.)
stances extracted from cell-free rumen fluid of cattle possess some of the classical properties of endotoxin found in such gram-negative bacteria as Eschericbia coil (Mullenax et al., 1966; Nagaraja et al., 1976). Having demonstrated that cell-free rumen fluid exhibits certain biological characteristics similar to endotoxins of gram-negative bacteria, we (Nagaraja et al., 1977) observed a quantitative difference in concentration of endotoxin in rumen fluids of cattle fed hay or grain. This report deals with a method to assay free endotoxin in rumen fluid of cattle based on the lethal effect on mice of cell-free rumen fluid potentiated with actinomycin D. Also, after obtaining evidence to confirm that the lethal agent was endotoxin we determined endotoxin concentrations in rumen fluid of cattle fed hay, grain or pasture. MATERIALS AND METHODS
INTRODUCTION
Effect o f lleat on Lethality o f Cell-free R u m e n Iquid. A rumen-fistulated adult Jersey cow was used as a source of rumen fluid. The cow was fed daily 5.44 kg of a 14% crude protein grain ration containing a mixture of sorghum grain (IRN 4-04-378), corn (IRN 4-02-931), soybean meal (IRN 5-04-004), Starea 2, dicalcium phosphate (1RN 6-01-080), salt (IRN 6-04-152), and a vitamin A and D supplement. Rumen fluid was obtained between 3 and 6 hr post feeding and immediately strained through four layers of cheesecloth. The strained tureen fluid was centrifuged at 450 g for 5 minutes. The supernatant containing bacteria was centrifuged at 25,000 g for 25 minutes. The resulting supernatant was determined cell-free by microscopic examination ~Contribution No. 78-24j, Department of Animal Sciences and Industry, Division of Biology (Micro- before being assayed. biology Section) and Veterinary Diagnostic LaboraRandom bred albino mice (20 to 25 g) of tory, Kansas Agricultural Experiment Station, Manhatboth sexes were fed a commercial stock diet tan. 2Starea is an extrusion processed mixture of grain (Purina Laboratory Chow, Ralston Purina Co., St. Louis, MO). Feed and water were provided and urea (registered trademark 860255, U.S. Patent No. 364.2489). ad libitum. Serial twofold dilutions of cell-free 1759 Rumen flora contains many gram-negative bacteria, a suspected source of endotoxin. Endotoxins may play an important role in the pathogenesis of certain diet-induced diseases like lactic acidosis, bloat and the sudden death syndrome (Dougherty and Cello, 1949, 1953; Mullenax et al., 1966; Dougherty et al., 1975; Dougherty, 1976; l[uber, 1976; Nagaraja et al., 1978). To produce disease, rumen bacterial endotoxins must be released from the bacteria and absorbed from the gastro-intestinal tract into the system. Therefore, free endotoxins should be detectable in cell-free rumen fluid. Sub-
JOURNAL OF ANIMAL SCIENCE, Vol. 46, No. 6, 1978 Downloaded from https://academic.oup.com/jas/article-abstract/46/6/1759/4699315 by guest on 09 April 2018
1760
NAGARAJA ET AL.
rumen fluid (minimum of four dilutions) were made in pyrogen-free saline and injected into groups of five mice each. This process was repeated a minimum of three times. The size of inoculum per mouse was .5 ml mixed with 20 gig of actinomycin D (Am D C o s m o g e n | Lots 1203T or 0724V. Merck Sharp and Dohme, West Point, PA). All injections were given intraperitoneally with sterile disposable syringes. Deaths were recorded up to 2 days. The LDs 0 was calculated by the Probit procedure with a modified Gauss-Newton logarithm to estimate the maximum likelihood of estimates of the intercept, slope and threshold response rate of the biological assay data (Finney, 11971). Cell-free rumen fluid was heated in a boiling water bath maintained at 100 C for 30, 180 or 360 minutes. Also, another sample was autoclaved (121 C, 15 psi for 20 min). The LDs 0 of the heated cell-free rumen fluid potentiated with Am D was estimated, and the change in LDs0 from the control was calculated.
F4"fect o f Acid Hydrolysis on Lethality o f Cell-]tee Rumen Fluid. One hundred ml of cell-free rumen fluid was treated with an equal volume of .2 N acetic acid. The mixture was kept in a boiling water bath for 30, 60, or 120 minutes. When removed from the bath, the mixture was quickly cooled to room temperature and neutralized with 10 N NaOH to the initial pH of rumen fluid. A control was prepared by adding acetic acid to cell-free rumen fluid and then neutralizing (without boiling) with 10 N NaOH to original pH. The neutralized heated or unheated cell-free rumen fluids were then concentrated in vacuum to obtain the original volume for the LDs 0 assay in mice.
Effect o f Alkaline tlydrolysis on Letbality of Cell-free Rumen Fluid. Cell-free rumen fluid was treated with .1 N NaOH and incubated at room temperature. At 3 or 6 or 24 hr, the NaOH-treated rumen fluid was neutralized with acetic acid to the initial pH and concentrated in vacuum to obtain the original volume. The control sample was prepared similarly by neutralizing NaOH-treated cell-free rumen fluid with acetic acid at 0 hours. The LDs0 was estimated in mice, and change in LDs0 from the control was calculated.
Protective Effect o f Cortisone on Lethality o f Cell-free Rumen Fluid. Groups of mice were injected subcutaneously with 5 mg (per mouse)
of cortisone (Cortone| acetate Merck, Sharp and Dohme, West Point, PA). The mouse LDs0 of cell-free rumen fluid was determined at 6 or 1 hr before, at the same time, and at 1 or 6 hr after injecting cortisone acetate. A control group received no cortisone acetate.
Lethal Effect o f Cell-free Rumen Fluid on Mice Made Tolerant to E. coil Endotoxin. The test was according to the procedure of Albizo and Surgallo (1970). One microgram of E. coli endotoxin (0111:B4 Difco Laboratories, Detroit, MI) contained in .1 ml of sterile pyrogenfree saline was injected intraperitoneally into each mouse (weight 20 to 25 g) on day one of the experiment. After 72 hr each mouse was injected with 10 gig of the same endotoxin. Also, each mouse in the control group (containing the same number of mice) was injected intraperitoneally with .1 ml of pyrogen-free saline on day one and .5 ml in 72 hours. On the 5th day, groups of control and test mice were injected with E. coil or Salmonella typhosa (0901 Difco) endotoxin with or without Am D or cell-free rumen fluid (with Am D) from cattle fed hay or grain. The dosages used are shown in table 5. Deaths were recorded up to 48 hours.
Concentration o f Free Endotoxin in Rumen Fluid from Cattle Fed All Hay or All Grain or ltigh-concentrate Ration. Two rumen-fistulated adult Jersey cows were used. One was fed daily 5.44 kg alfalfa hay (1RN 1-00-063) and the other received daily 5.44 kg of a 14% crude protein grain ration similar to that used previously. Rumen fluid samples were obtained at 0, 3, 6, 12 and 24 hr post feeding. The LDs 0 of cell-free rumen fluid potentiated with 20 gig Am D was determined in mice. The concentration of endotoxin in rumen fluid expressed as rumen bacterial endotoxin activity was calculated on the basis that LDs 0 of cell-free rumen fluid should contain .86 gig of endotoxin which is the established LDs0 of rumen bacterial endotoxin (Nagaraja et al., 1978). Total bacterial numbers in rumen fluid were determined by diluting samples with 10% formal saline and counting in a Petroff-Hausser bacterial counting cl~amber (C. A. Hausser and Son, Philadelphia, PA). Percentages of gram-negative and grampositive bacteria were estimated by counting a minimum of 1,000 cells fr~)m a gram-stained smear. The smear was made by spreading evenly 5 gil of diluted rumen fluid on an area of 1 cm 2 (Warner, 1962). All counts were done in duplicate and were averaged.
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ENDOTOXIN IN CATTLE RUMEN FLUID Also, rumen fluid samples were obtained 6 hr post feeding from five rumen-fistulated cows and five heifers adapted to an all roughage ration of alfalfa hay; from five steers (400 to 450 kg) fed a high-concentrate low roughage ration for more than 100 days; and from five steers (375 to 400 kg) fed an all concentrate ration for more than 100 days. The steers on the high-concentrate low-roughage ration were fed twice daily ad libitum quantities (as-fed basis) a ration of 24.0% sorghum silage (31.8% dry matter), 70.2% rolled sorghum grain, 4.31% soybean meal, .12% urea, (IRN 5-05-070),. 37% limestone (IRN 6-02-632), . 3% dicalcium phosphate, .5% salt, .05% trace mineral (Z-5 Calcium Carbonate Co., Quincy, IL), .04% aureomycin (22.2 g/kg), .01% vitamin A (30,000 IU/g). The steers fed an all concentrate ration were fed 8.33 kg (divided into two feedings) of the following: 62% rolled sorghum grain, 25% cracked wheat (IRN 4-05-268), 10% Starea, 2% dicalcium phosphate, .5% trace mineralized salt, and .5% vitamin A and D supplement (1,000,000 units of A and D per kilogram supplement). The LDs0 of the cell-free rumen fluid, endotoxin activity, total bacterial number, percentages of gram-negative and positive bacteria were determined as before. RESULTS AND DISCUSSION
Effect of Heat on Lethality o f Cell-free Rumen Fluid. Unheated cell-free rumen fluid was lethal to mice (LDs 0 = .054 ml, table 1). Heating cell-free rumen fluid at 100 C for 30 rain did n o t change the LDs0. However, pro-
1761
longed heating (3 or 6 hr) reduced the toxicity of cell-free rumen fluid by a factor of 1.7 and 3.0, respectively. Autoclaving cell-free rumen fluid caused a small decrease in toxicity. Therefore, the toxic principle responsible for the death of mice apparently is resistant to heat (at least for 30 min). Extreme heat tolerance was one of the earliest methods used to distinguish endotoxins from other bacterial toxins, but no uniformity has been noted in the m i n i m u m temperature needed to destroy endotoxin (Milner et al., 1971). Neter et al. (1956) observed that heating Salmonella abortus equi endotoxin at 100 C for 2.5 hr did not alter its toxicity.
Effect o f Acid Hydrolysis on Lethality of Cell-free Rumen Fluid. The toxicity of cell-free rumen fluid boiled with .2 N acetic acid decreased progressively with time (table 2). The LDs0 increased about two and a half times after hydrolyzing for 30 min and almost four times after 2 hours. Mild acid apparently inactivates the toxic principle in rumen fluid. The loss of biological activity (including mouse lethality) of endotoxin when exposed to acid at elevated temperature has been shown (Sultzer, 1971). Tal and Goebel (1950) showed that Shigella endotoxin treated with 1% acetic acid at 100 C reduced mouse lethality by four times within 60 minutes. Haskins et al. (1961), who studied in detail the effects of acid hydrolysis on biological activities of Salmonella enteritidis endotoxin, observed that within 15 min the lethality of the endotoxin in mice decreased dramatically. In our study, acid hydrolysis of the cell-free rumen fluid for 2 hr did not render it completely nontoxic.
TABLE 1. EFFECT OF HEAT ON LETHALITY OF CELL-FREE RUMEN FLUID PLUS ACTINOMYCIND ON MICEa
Heat treatment
Duration of treatment
None (control) 100 c 100 C 100 C Autoclaved
(min) 0 30 180 360 20
LDs o
Increase in LD 5o
(ml) .054 +- .004b .053 +- .011
...
.093 + .034*
117x
.164 + .028* .089 -+.018"
3.0x 1.6x
aAverage of three experiments. bMean + standard error.
*Significantly different from control (P
