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Electrophoretic Separation of Serum Proteins and Lipoproteins of Young Turkeys Infected with Eimeria meleagrimitis or Eimeria adenoeides
Electrophoretic Separation of Serum Proteins and Lipoproteins of Young Turkeys Infected with Eimeria meleagrimitis or Eimeria adenoeides P. C. AUGUSTINE Animal Parasitology Institute, Beltsville Agricultural Research Center — East, Building 1120, Beltsville, Maryland 20705 (Received for publication November 11, 1984)
1985 Poultry Science 64:1644-1648
INTRODUCTION T o t a l serum p r o t e i n of t u r k e y poults decreased significantly b y Day 7 postinoculation (PI) w i t h severe Eimeria infection (Augustine and T h o m a s , 1979, 1981). However, t h e e x t e n t of t h e decrease was n o t always predictable from t h e severity of t h e infection. Changes in serum protein levels indicate conditions t h a t alter t h e balance b e t w e e n synthesis and utilization of protein. In chickens, these alterations are reflected in shifts in t h e individual protein fractions t h a t m a k e u p t h e t o t a l serum p r o t e i n (Ruff and Augustine, 1 9 8 2 ) . T o d e t e r m i n e if such shifts also occur in t u r k e y s , serum proteins and plasma lipoproteins of p o u l t s m o d e r a t e l y infected with Eimeria meleagrimitis or E. adenoeides were separated electrophoretically and c o m p a r e d with t h o s e of fully-fed, uninoculated controls. Additionally, because anorexia is characteristic of Eimeria infections, t h e serum protein profiles of pair-fed, u n i n o c u l a t e d control poults were also e x a m i n e d .
MATERIALS AND METHODS D i a m o n d Hybrid female p o u l t s were obtained at 1 day of age, h o u s e d in wire-floored
cages, and allowed t o feed ad libitum. At 1.5 or 2.5 w e e k s of age, t h e poults were orally inoculated with either E. adenoeides or E. meleagrimitis oocysts. On Days 7 or 8 PI, t h e p o u l t s were weighed and bled b y cardiac p u n c t u r e according t o t h e design of t h e e x p e r i m e n t . Experiment 1. Fifteen 2.5-week-old p o u l t s were inoculated with 5 X 1 0 5 E. adenoeides oocysts. On D a y 7 PI, serum was recovered. Fifteen u n i n o c u l a t e d controls were fully fed. Experiment 2. Twelve 1.5-week-old p o u l t s received 1 X 1 0 s E. meleagrimitis oocysts. On Day 7 PI, serum was recovered and pooled within groups, 3 samples per pool. Twelve u n inoculated controls were fully fed. Experiment 3. Six 2.5-week-old poults w e r e inoculated with 1 x 1 0 s E. adenoeides o o c y s t s . On Day 8 PI, serum was recovered. Six u n inoculated controls were fully fed. Experiment 4. Twelve 1.5-week-old p o u l t s were given 1 X 1 0 5 E. meleagrimitis oocysts. On Day 8 PI, serum was recovered and pooled within groups, 3 samples per pool. Twelve uninoculated controls were fully fed. Experiment 5. T h i r t y 2.5-week-old p o u l t s were u s e d ; 6 were allowed t o feed ad libitum (fully-fed controls), 6 were inoculated w i t h 2
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ABSTRACT Electrophoretic separation of serum proteins of Eimeria-infected turkeys showed five major fractions with significant decreases in albumin levels and significant increases in the a2and -/-globulins as compared with controls. The a r and (3-globulin levels were similar to those of the controls. Significant differences between infected turkeys and uninfected pair-fed controls suggested that protein changes were not solely due to the anorexia associated with Eimeria infections. The increase in a 2 -globulins was not due to an increase in ceruloplasmin. Indirect fluorescent antibody tests using serum from infected poults as an antibody source were positive, indicating that at least part of the increase in the 7-globuIin fraction was due to the production of immunoglobulins. Electrophoretic separation of plasma lipoproteins showed three to four major bands with a sharp decrease in the portomicrons among infected poults. There was no increase in the pre-|3lipoproteins (which have the same electrophoretic mobility as the a 2 -globulins), indicating that the increase in a2-globulins was not due to an increase in the lipoprotein part of this fraction. (Key words: turkeys, coccidiosis, serum proteins, lipoproteins, electrophoresis)
PROTEINS, LIPOPROTEINS, AND COCCIDIOSIS RESULTS AND DISCUSSION
Electrophoretic separation of serum from 3-week-old turkey poults demonstrated five major protein fractions that comigrated with the albumin and a^, a 2 -, /J-, and 7-globulins of human serum (Fig. 1). Serum from uninfected turkeys contained an average of 64% albumin, 15% a r plus a 2 -globulins, 14% |3-globulins, and 7% 7-globulins. This distribution is similar to that reported for 4- to 6-month-old turkeys (Lynch and Stafseth, 1953). The changes in the total serum protein levels in Eimeria-infected turkeys appears to be due to decreases in the albumin fraction combined with increases in the a2~ and 7-globulin fractions. The changes in fractions occurred in both 1.5- and 2.5-week-old poults. The shifts caused significant decreases in the albumin/globulin ratio from 1.97 ± .09 in control turkeys to 1.09 ± .05 and 1.23 ± .19 in poults infected with E. adenoeides and E. meleagrimitis, respectively. Albumin was invariably decreased even in moderate infections, but the decrease was usually not significant until Day 8 PI. In the present study, on Day 7 PI, poults infected with E. adenoeides or E. meleagrimitis oocysts showed weight gains of only 62 and 40% of that of the controls, respectively (Table 1). Albumin in
Alb
e c - i o c - 2 ptf
FIG. 1. Electrophoretic separation of serum proteins of uninfected 3-week-old turkey poults showing albumin (alb), a,-, a2-, (3-, and 7-globulin fractions. Arrow indicates point of application of serum.
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x 10 E. adenoeides oocysts, 6 were inoculated with 1.5 X 10 5 E. meleagrimitis oocysts, and 6 were pair-fed with each of the inoculated groups (pair-fed controls). Each day, the inoculated groups and their pair-fed counterparts were restricted to the amount of feed consumed the previous day by turkeys infected 1 day earlier than the birds in the experiment. On Day 8 PI, serum was recovered. Experiment 6. Nine uninfected poults were bled. Serum samples were divided in half, leaving some red blood cells in half of each sample. Both halves were frozen, thawed, and centrifuged before electrophoresis. Experiments 7 and 8. In each experiment, 4 of the 2.5-week-old turkeys were inoculated with 1.5 x 10 5 E. adenoeides oocysts, 4 with 1.5 X 10 s E. meleagrimitis oocysts, and 8 were uninoculated, fully-fed controls. On Day 8 PI, plasma was recovered and examined as individual samples and as pools within groups. For serum protein determinations (Experiments 1 through 6), no anticoagulant was used; the blood was allowed to clot at room temperature, centrifuged, and the serum was recovered. For lipoprotein determinations (Experiments 7 and 8), blood was collected in tubes containing 100 ng ethylenediaminetetraacetate (EDTA) and kept on ice until the plasma was recovered. To aid in identification of fractions, a sample of human serum was electrophoresed along with the samples of turkey serum. Total plasma or serum protein was determined by dye binding (Bio-Rad Laboratories, Richmond, CA). Protein fractions were separated on cellulose acetate strips at 225 V for 30 min using a Sepra Tek system (Gelman Instrument Company, Ann Arbor, MI). Albumin and globulins were stained with Ponceau S, and lipoproteins with Oil Red 0, according to procedures outlined in the Gelman Technical Bulletin No. 28 (1978). All strips were scanned on a Corning 760 fluorometer/densitometer (Corning Glass Works, Medfield, ME) at a wavelength of 520 nm. Albumin and globulin fractions were quantitated by multiplying the percent protein in each fraction by the grams of protein per 100 ml of serum. Ceruloplasmin was measured according to a modification of the method of Smith and Wright (1974). Differences between treatment groups were tested by analysis of variance, and where applicable, Duncan's multiple range procedure. Probabilities equal to or less than .05 were considered statistically significant.
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AUGUSTINE TABLE 1. Serum protein measurements of Eimeria-infected and control turkeys on Day 7 postinoculation Experiment 2 2
Experiment l 1 Observation Number Inoculum Gain, g Total protein, g/100 ml Albumin, g/100 ml a2 -Globulin, g/100 ml (3-Globulin, g/100 ml 7-Globulin, g/100 ml
Control
E. adenoeides
Control
15 0 229 ± 9 4.5 ± .2 2.6 ± .1 .6± .1 .8 ± .1 .2+ .02
15 5 X 10 s 92 ± 7* 4.7+ .1 2.3+ .1 .8± . 1 * .9± .1 .3± .04*
4 0 146 ± 7 4.4 ± .2 3.0+ .2 .4± .1 .6± .1 .2 + .02
E. meleagrimitis 3 1 X 10 s 59 ± 10* 4.9 ± .1 2.6 ± .1 .7 ± .1 .8+ .1 .4 + .04
1
In Experiment 1, data expressed as mean ± standard error of individual samples. In Experiment 2, data expressed as mean ± standard error of pooled samples (3 sera/pool). 'Significantly different (P<.05) from control mean within experiments.
2
that the development of the parasite in the intestine may be as important as anorexia in the decrease in serum albumin. This proposal is supported by earlier findings that total plasma protein in uninfected turkeys was significantly decreased only after 72 hr of complete starvation (Augustine, 1982). Two globulin fractions, those comigrating with the a2- and 7-globulins of human serum, were significantly increased in poults inoculated with either E. adenoeides or E. meleagrimitis; the ail- and /J-globulins were unchanged. The increase in a 2 -globulins was significant on Days 7 and 8 PI with E. adenoeides infection and on Day 8 PI with E. meleagrimitis (Tables 1 and
TABLE 2. Serum protein measurements of Eimeria-infected and control turkeys on Day 8 postinoculation Experiment 4 2
Experiment 3 1 Observation Number Inoculum Gain, g Total protein, g/100 ml Albumin, g/100 ml a2 -Globulin, g/100 ml (3-Globulin, g/100 ml 7-Globulin, g/100 ml 1
Control
E. adenoeides
Control
6 0 237 ± 10 4.8 ± .4 .1 2.9 + .4± .04 .6± .1 .3 ± .04
6 1 X 10 s 146 + 5* 3.6+ .3* 2.5 ± .2* .6± . 1 * .7 ± .1 .5 ± . 1 *
4 0 179 ± 10 4.7 + .2 2.8 ± .2 .5+ .1 .7 ± .1 .2 ± .02
In Experiment 3, data expressed as mean ± standard error of individual samples. In Experiment 4, data expressed as mean + standard error of pooled samples (3 sera/pool). * Significantly different (P=S.05) from control mean within experiments.
2
E. meleagrimitis 4 1 X 10 5 101 ± 5* 4.5 + .2 2.2+ . 1 * .8± .2* 1.0+ .3 .5 ± . 1 *
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both groups of infected poults was slightly decreased. By Day 8 PI, albumin was significantly decreased by both species (Table 2), even though the weight gain of the E. meleagrimitis-iniected poults was better than that at 7 PI. Albumin is thought to act as a protein source during times of subnormal feed intake (Sturkie, 1976). Feed intake of poults infected with these two species of Eimeria was low at around Day 8 PI (Augustine and Thomas, 1981), and therefore, the anorexia associated with the infection may have contributed to the drop in albumin. However, differences in albumin levels between infected poults and uninoculated pair-fed controls (Table 3) suggest
PROTEINS, LIPOPROTEINS, AND COCCIDIOSIS
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TABLE 3. Serum protein measurements of Eimeria-infected, fully-fed, and pair-fed control turkeys on Day 8 postinoculation E. Fully-fed controls
Observation Number Inoculum Gain, g Total protein, g/100 ml Albumin, g/100 ml a2-Globulin, g/100 ml /3-Globulin, g/100 ml 7-Globulin, g/100 ml
6 0 213 ± 9 4 . 8 ± .3 3.0 ± .2 . 4 ± .04 .7 ± .04 .2 ± . 0 1
E. m eleagrimitis
adenoeides
Pair-fed controls
Inoculated
6 0 68 ± 7* 4 . 8 + .2 3.1 ± .2 .5 ± .04 . 7 + .1 .1 ± .01
4 2 X 10s 11 ± 1 5 * 4.2 ± .3 2.0+ .2* .6 ± .01* .7 ± .1 .4 ± .02*
Pair-fed controls 6 0 108 4.9 3.3 .5 .7 .2
± 6* ± .01 ± .2 ± .1 ± .1 + .02
Inoculated 5 1.5 X 1 0 s 60 + 20* 5.1 ± .01 2.6+ .1 .9 ± .01* .9 ± .04 .4+ .01*
1
2). The lack of significance on Day 7 PI in E. meleagrimitis infection is probably due to the relatively low members of observations (pools rather than individual samples). Generally speaking, c^-globulins are elevated in conditions involving acute inflammatory processes, tissue destruction, necrosis, and altered vascular permeability (Kelsey, 1969). These conditions have all been mentioned as part of the pathophysiology of Eimeria infection. The specific proteins that increased to produce the increase in the QJ2-globulin fraction have not been identified. Serum samples from three experiments showed no increase in plasma ceruloplasmin on Day 8 PI (Mark Richards, personal communication). Furthermore, electrophoretic separation of plasma lipoproteins revealed no increase in the pre-/? fraction which has the same electrophoretic mobility as the
a 2 -globulins. Therefore, the significant increase in a 2 -globulins with Eimeria infection was apparently not due to a marked increase in the lipoproteins associated with the fraction. The significant increase in y-globulins was seen only among the Eimeria-infected poults. Indirect fluorescent antibody (IFA) tests using Day 8 PI serum as an antibody source were positive, indicating that the increase was due, at least in part, to an increase in immunoglobulins. Little change was seen in a2~ and 7-globulins among uninoculated birds that were pair-fed with Eimeria-inocuhted ones (Table 3). This indicates that the changes in the globulin, like the changes in the albumin, are due more to the infection than to the anorexia associated with the infection. Furthermore, hemolysis of serum samples had no effect on either the separation or amounts of the protein fractions (Table 4).
TABLE 4. Serum protein measurements ofhemolyzed vs. unhemolyzed serum (Experiment 6)
1
Observation
Unhemolyzed
Hemolyzed
Number Total protein, g/100 ml Albumin, g/100 ml a2 -Globulin, g/100 ml (3-Globulin, g/100 ml 7-Globulin, g/100 ml
9 5.5 ± . 4 ' 3.27 .63 .79 .29
9 5.7 + . l 1 3.43 .62 .77 .32
Mean of 9 individual samples. Half of each sample was hemolyzed; half was protected from hemolysis.
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Data expressed as mean ± standard error of pooled samples (3 sera/pool). •Significantly different (P<.05) from fully-fed control levels.
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AUGUSTINE fied in all samples b u t t h e pre-)3-fraction was n o t always present. T h e r e was n o correlation b e t w e e n infection and presence or absence of t h e pre-j3-lipoproteins. E x c e p t for m o r e p o r t o microns, t h e r e were n o consistent differences b e t w e e n lipoproteins of control and infected poults. This contrasts w i t h m a r k e d differences observed b e t w e e n coccidia-infected and control chickens at various intervals PI (Patricia Allen, personal c o m m u n i c a t i o n ) . It m a y b e t h a t coccidial infection p r o d u c e d little effect o n t h e lipoprotein profiles in t u r k e y s or t h a t if t h e effect occurred earlier, it was t r a n s i t o r y and was n o t discernible on Day 8 PI.
T h e a u t h o r s t h a n k Pauldo Smith, Lourdes Carson, a n d Freeda Isaac for excellent technical assistance, Mark Richards for t h e ceruloplasmin assays, and G a r y Peck for assistance in preparing t h e figures. Special t h a n k s is e x t e n d e d t o Allen Edgar for t h e original isolate of t h e Eimeria species.
REFERENCES
FIG. 2. Electrophoretic separation of plasma lipoproteins of control (A), E. meleagrimitis-infected (B), and E. adenoeides-infected (C) 3-week-old turkeys. Four fractions are shown: portomicrons (PM), /?-, pre-0 (p/3)-, and a-lipoproteins. Arrow indicates point of application of plasma.
Therefore, hemolysis t h a t often occurs as severely-infected p o u l t s are bled did n o t interfere with t h e separation or q u a n t i t a t i o n of serum p r o t e i n fractions. E l e c t r o p h o r e t i c separation and staining with Oil R e d 0 showed four lipoprotein fractions: p o r t o m i c r o n s , /}-, pre-/?-, and a-lipoproteins (Fig. 2). Because t h e poults were n o t fasted before blood samples were t a k e n , p o r t o m i c r o n s were present in b o t h controls and infected plasma, b u t t h e y were m o r e c o n c e n t r a t e d in t h e controls. Beta and a-lipoproteins were identi-
Augustine, P. C , 1982. Effect of feed and water deprivation on organ and blood characteristics of young turkeys. Poultry Sci. 61:796-799. Augustine, P. C , and O. P. Thomas, 1979. Eimeria meleagrimitis in young turkeys: effects on weight, blood, and organ parameters. Avian Dis. 24:854-862. Augustine, P. C , and O. P. Thomas, 1981. Effect of time on response to Eimeria adenoeides and Eimeria meleagrimitis infection in young turkeys. Avian Dis. 25:366-373. Gelman Instrument Company, 1978. Lipoprotein electrophoresis system. Tech. Bull. 28. Kelsey, R. L., 1969. Interpretation of routine electrophoretic patterns of serum proteins in health and disease. In Gelman Manual of Clinical Electrophoresis. Ann Arbor, MI. Lynch, J. E., and H. J. Stafseth, 1953. Electrophoretic studies on the serum proteins of turkeys. The composition of normal turkey serum. Poultry Sci. 32:1068-1073. Ruff, M. D., and P. C. Augustine, 1982. Effects of coccidiosis on the electrophoretic pattern of serum proteins in chickens. J. Parasitol. 68:107— 111. Smith, B.S.W., and H. Wright, 1974. Improved manual and automated procedures for estimation of ceruloplasmin oxidase activity. Clin. Chim. Acta 50:359-366. Sturkie, P. D., 1976. Avian Physiology. P. D. Sturkie, ed. Springer-Verlag, New York, NY.
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ACKNOWLEDGMENTS
1985 Poultry Science 64:1644-1648
INTRODUCTION T o t a l serum p r o t e i n of t u r k e y poults decreased significantly b y Day 7 postinoculation (PI) w i t h severe Eimeria infection (Augustine and T h o m a s , 1979, 1981). However, t h e e x t e n t of t h e decrease was n o t always predictable from t h e severity of t h e infection. Changes in serum protein levels indicate conditions t h a t alter t h e balance b e t w e e n synthesis and utilization of protein. In chickens, these alterations are reflected in shifts in t h e individual protein fractions t h a t m a k e u p t h e t o t a l serum p r o t e i n (Ruff and Augustine, 1 9 8 2 ) . T o d e t e r m i n e if such shifts also occur in t u r k e y s , serum proteins and plasma lipoproteins of p o u l t s m o d e r a t e l y infected with Eimeria meleagrimitis or E. adenoeides were separated electrophoretically and c o m p a r e d with t h o s e of fully-fed, uninoculated controls. Additionally, because anorexia is characteristic of Eimeria infections, t h e serum protein profiles of pair-fed, u n i n o c u l a t e d control poults were also e x a m i n e d .
MATERIALS AND METHODS D i a m o n d Hybrid female p o u l t s were obtained at 1 day of age, h o u s e d in wire-floored
cages, and allowed t o feed ad libitum. At 1.5 or 2.5 w e e k s of age, t h e poults were orally inoculated with either E. adenoeides or E. meleagrimitis oocysts. On Days 7 or 8 PI, t h e p o u l t s were weighed and bled b y cardiac p u n c t u r e according t o t h e design of t h e e x p e r i m e n t . Experiment 1. Fifteen 2.5-week-old p o u l t s were inoculated with 5 X 1 0 5 E. adenoeides oocysts. On D a y 7 PI, serum was recovered. Fifteen u n i n o c u l a t e d controls were fully fed. Experiment 2. Twelve 1.5-week-old p o u l t s received 1 X 1 0 s E. meleagrimitis oocysts. On Day 7 PI, serum was recovered and pooled within groups, 3 samples per pool. Twelve u n inoculated controls were fully fed. Experiment 3. Six 2.5-week-old poults w e r e inoculated with 1 x 1 0 s E. adenoeides o o c y s t s . On Day 8 PI, serum was recovered. Six u n inoculated controls were fully fed. Experiment 4. Twelve 1.5-week-old p o u l t s were given 1 X 1 0 5 E. meleagrimitis oocysts. On Day 8 PI, serum was recovered and pooled within groups, 3 samples per pool. Twelve uninoculated controls were fully fed. Experiment 5. T h i r t y 2.5-week-old p o u l t s were u s e d ; 6 were allowed t o feed ad libitum (fully-fed controls), 6 were inoculated w i t h 2
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ABSTRACT Electrophoretic separation of serum proteins of Eimeria-infected turkeys showed five major fractions with significant decreases in albumin levels and significant increases in the a2and -/-globulins as compared with controls. The a r and (3-globulin levels were similar to those of the controls. Significant differences between infected turkeys and uninfected pair-fed controls suggested that protein changes were not solely due to the anorexia associated with Eimeria infections. The increase in a 2 -globulins was not due to an increase in ceruloplasmin. Indirect fluorescent antibody tests using serum from infected poults as an antibody source were positive, indicating that at least part of the increase in the 7-globuIin fraction was due to the production of immunoglobulins. Electrophoretic separation of plasma lipoproteins showed three to four major bands with a sharp decrease in the portomicrons among infected poults. There was no increase in the pre-|3lipoproteins (which have the same electrophoretic mobility as the a 2 -globulins), indicating that the increase in a2-globulins was not due to an increase in the lipoprotein part of this fraction. (Key words: turkeys, coccidiosis, serum proteins, lipoproteins, electrophoresis)
PROTEINS, LIPOPROTEINS, AND COCCIDIOSIS RESULTS AND DISCUSSION
Electrophoretic separation of serum from 3-week-old turkey poults demonstrated five major protein fractions that comigrated with the albumin and a^, a 2 -, /J-, and 7-globulins of human serum (Fig. 1). Serum from uninfected turkeys contained an average of 64% albumin, 15% a r plus a 2 -globulins, 14% |3-globulins, and 7% 7-globulins. This distribution is similar to that reported for 4- to 6-month-old turkeys (Lynch and Stafseth, 1953). The changes in the total serum protein levels in Eimeria-infected turkeys appears to be due to decreases in the albumin fraction combined with increases in the a2~ and 7-globulin fractions. The changes in fractions occurred in both 1.5- and 2.5-week-old poults. The shifts caused significant decreases in the albumin/globulin ratio from 1.97 ± .09 in control turkeys to 1.09 ± .05 and 1.23 ± .19 in poults infected with E. adenoeides and E. meleagrimitis, respectively. Albumin was invariably decreased even in moderate infections, but the decrease was usually not significant until Day 8 PI. In the present study, on Day 7 PI, poults infected with E. adenoeides or E. meleagrimitis oocysts showed weight gains of only 62 and 40% of that of the controls, respectively (Table 1). Albumin in
Alb
e c - i o c - 2 ptf
FIG. 1. Electrophoretic separation of serum proteins of uninfected 3-week-old turkey poults showing albumin (alb), a,-, a2-, (3-, and 7-globulin fractions. Arrow indicates point of application of serum.
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x 10 E. adenoeides oocysts, 6 were inoculated with 1.5 X 10 5 E. meleagrimitis oocysts, and 6 were pair-fed with each of the inoculated groups (pair-fed controls). Each day, the inoculated groups and their pair-fed counterparts were restricted to the amount of feed consumed the previous day by turkeys infected 1 day earlier than the birds in the experiment. On Day 8 PI, serum was recovered. Experiment 6. Nine uninfected poults were bled. Serum samples were divided in half, leaving some red blood cells in half of each sample. Both halves were frozen, thawed, and centrifuged before electrophoresis. Experiments 7 and 8. In each experiment, 4 of the 2.5-week-old turkeys were inoculated with 1.5 x 10 5 E. adenoeides oocysts, 4 with 1.5 X 10 s E. meleagrimitis oocysts, and 8 were uninoculated, fully-fed controls. On Day 8 PI, plasma was recovered and examined as individual samples and as pools within groups. For serum protein determinations (Experiments 1 through 6), no anticoagulant was used; the blood was allowed to clot at room temperature, centrifuged, and the serum was recovered. For lipoprotein determinations (Experiments 7 and 8), blood was collected in tubes containing 100 ng ethylenediaminetetraacetate (EDTA) and kept on ice until the plasma was recovered. To aid in identification of fractions, a sample of human serum was electrophoresed along with the samples of turkey serum. Total plasma or serum protein was determined by dye binding (Bio-Rad Laboratories, Richmond, CA). Protein fractions were separated on cellulose acetate strips at 225 V for 30 min using a Sepra Tek system (Gelman Instrument Company, Ann Arbor, MI). Albumin and globulins were stained with Ponceau S, and lipoproteins with Oil Red 0, according to procedures outlined in the Gelman Technical Bulletin No. 28 (1978). All strips were scanned on a Corning 760 fluorometer/densitometer (Corning Glass Works, Medfield, ME) at a wavelength of 520 nm. Albumin and globulin fractions were quantitated by multiplying the percent protein in each fraction by the grams of protein per 100 ml of serum. Ceruloplasmin was measured according to a modification of the method of Smith and Wright (1974). Differences between treatment groups were tested by analysis of variance, and where applicable, Duncan's multiple range procedure. Probabilities equal to or less than .05 were considered statistically significant.
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AUGUSTINE TABLE 1. Serum protein measurements of Eimeria-infected and control turkeys on Day 7 postinoculation Experiment 2 2
Experiment l 1 Observation Number Inoculum Gain, g Total protein, g/100 ml Albumin, g/100 ml a2 -Globulin, g/100 ml (3-Globulin, g/100 ml 7-Globulin, g/100 ml
Control
E. adenoeides
Control
15 0 229 ± 9 4.5 ± .2 2.6 ± .1 .6± .1 .8 ± .1 .2+ .02
15 5 X 10 s 92 ± 7* 4.7+ .1 2.3+ .1 .8± . 1 * .9± .1 .3± .04*
4 0 146 ± 7 4.4 ± .2 3.0+ .2 .4± .1 .6± .1 .2 + .02
E. meleagrimitis 3 1 X 10 s 59 ± 10* 4.9 ± .1 2.6 ± .1 .7 ± .1 .8+ .1 .4 + .04
1
In Experiment 1, data expressed as mean ± standard error of individual samples. In Experiment 2, data expressed as mean ± standard error of pooled samples (3 sera/pool). 'Significantly different (P<.05) from control mean within experiments.
2
that the development of the parasite in the intestine may be as important as anorexia in the decrease in serum albumin. This proposal is supported by earlier findings that total plasma protein in uninfected turkeys was significantly decreased only after 72 hr of complete starvation (Augustine, 1982). Two globulin fractions, those comigrating with the a2- and 7-globulins of human serum, were significantly increased in poults inoculated with either E. adenoeides or E. meleagrimitis; the ail- and /J-globulins were unchanged. The increase in a 2 -globulins was significant on Days 7 and 8 PI with E. adenoeides infection and on Day 8 PI with E. meleagrimitis (Tables 1 and
TABLE 2. Serum protein measurements of Eimeria-infected and control turkeys on Day 8 postinoculation Experiment 4 2
Experiment 3 1 Observation Number Inoculum Gain, g Total protein, g/100 ml Albumin, g/100 ml a2 -Globulin, g/100 ml (3-Globulin, g/100 ml 7-Globulin, g/100 ml 1
Control
E. adenoeides
Control
6 0 237 ± 10 4.8 ± .4 .1 2.9 + .4± .04 .6± .1 .3 ± .04
6 1 X 10 s 146 + 5* 3.6+ .3* 2.5 ± .2* .6± . 1 * .7 ± .1 .5 ± . 1 *
4 0 179 ± 10 4.7 + .2 2.8 ± .2 .5+ .1 .7 ± .1 .2 ± .02
In Experiment 3, data expressed as mean ± standard error of individual samples. In Experiment 4, data expressed as mean + standard error of pooled samples (3 sera/pool). * Significantly different (P=S.05) from control mean within experiments.
2
E. meleagrimitis 4 1 X 10 5 101 ± 5* 4.5 + .2 2.2+ . 1 * .8± .2* 1.0+ .3 .5 ± . 1 *
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both groups of infected poults was slightly decreased. By Day 8 PI, albumin was significantly decreased by both species (Table 2), even though the weight gain of the E. meleagrimitis-iniected poults was better than that at 7 PI. Albumin is thought to act as a protein source during times of subnormal feed intake (Sturkie, 1976). Feed intake of poults infected with these two species of Eimeria was low at around Day 8 PI (Augustine and Thomas, 1981), and therefore, the anorexia associated with the infection may have contributed to the drop in albumin. However, differences in albumin levels between infected poults and uninoculated pair-fed controls (Table 3) suggest
PROTEINS, LIPOPROTEINS, AND COCCIDIOSIS
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TABLE 3. Serum protein measurements of Eimeria-infected, fully-fed, and pair-fed control turkeys on Day 8 postinoculation E. Fully-fed controls
Observation Number Inoculum Gain, g Total protein, g/100 ml Albumin, g/100 ml a2-Globulin, g/100 ml /3-Globulin, g/100 ml 7-Globulin, g/100 ml
6 0 213 ± 9 4 . 8 ± .3 3.0 ± .2 . 4 ± .04 .7 ± .04 .2 ± . 0 1
E. m eleagrimitis
adenoeides
Pair-fed controls
Inoculated
6 0 68 ± 7* 4 . 8 + .2 3.1 ± .2 .5 ± .04 . 7 + .1 .1 ± .01
4 2 X 10s 11 ± 1 5 * 4.2 ± .3 2.0+ .2* .6 ± .01* .7 ± .1 .4 ± .02*
Pair-fed controls 6 0 108 4.9 3.3 .5 .7 .2
± 6* ± .01 ± .2 ± .1 ± .1 + .02
Inoculated 5 1.5 X 1 0 s 60 + 20* 5.1 ± .01 2.6+ .1 .9 ± .01* .9 ± .04 .4+ .01*
1
2). The lack of significance on Day 7 PI in E. meleagrimitis infection is probably due to the relatively low members of observations (pools rather than individual samples). Generally speaking, c^-globulins are elevated in conditions involving acute inflammatory processes, tissue destruction, necrosis, and altered vascular permeability (Kelsey, 1969). These conditions have all been mentioned as part of the pathophysiology of Eimeria infection. The specific proteins that increased to produce the increase in the QJ2-globulin fraction have not been identified. Serum samples from three experiments showed no increase in plasma ceruloplasmin on Day 8 PI (Mark Richards, personal communication). Furthermore, electrophoretic separation of plasma lipoproteins revealed no increase in the pre-/? fraction which has the same electrophoretic mobility as the
a 2 -globulins. Therefore, the significant increase in a 2 -globulins with Eimeria infection was apparently not due to a marked increase in the lipoproteins associated with the fraction. The significant increase in y-globulins was seen only among the Eimeria-infected poults. Indirect fluorescent antibody (IFA) tests using Day 8 PI serum as an antibody source were positive, indicating that the increase was due, at least in part, to an increase in immunoglobulins. Little change was seen in a2~ and 7-globulins among uninoculated birds that were pair-fed with Eimeria-inocuhted ones (Table 3). This indicates that the changes in the globulin, like the changes in the albumin, are due more to the infection than to the anorexia associated with the infection. Furthermore, hemolysis of serum samples had no effect on either the separation or amounts of the protein fractions (Table 4).
TABLE 4. Serum protein measurements ofhemolyzed vs. unhemolyzed serum (Experiment 6)
1
Observation
Unhemolyzed
Hemolyzed
Number Total protein, g/100 ml Albumin, g/100 ml a2 -Globulin, g/100 ml (3-Globulin, g/100 ml 7-Globulin, g/100 ml
9 5.5 ± . 4 ' 3.27 .63 .79 .29
9 5.7 + . l 1 3.43 .62 .77 .32
Mean of 9 individual samples. Half of each sample was hemolyzed; half was protected from hemolysis.
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Data expressed as mean ± standard error of pooled samples (3 sera/pool). •Significantly different (P<.05) from fully-fed control levels.
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AUGUSTINE fied in all samples b u t t h e pre-)3-fraction was n o t always present. T h e r e was n o correlation b e t w e e n infection and presence or absence of t h e pre-j3-lipoproteins. E x c e p t for m o r e p o r t o microns, t h e r e were n o consistent differences b e t w e e n lipoproteins of control and infected poults. This contrasts w i t h m a r k e d differences observed b e t w e e n coccidia-infected and control chickens at various intervals PI (Patricia Allen, personal c o m m u n i c a t i o n ) . It m a y b e t h a t coccidial infection p r o d u c e d little effect o n t h e lipoprotein profiles in t u r k e y s or t h a t if t h e effect occurred earlier, it was t r a n s i t o r y and was n o t discernible on Day 8 PI.
T h e a u t h o r s t h a n k Pauldo Smith, Lourdes Carson, a n d Freeda Isaac for excellent technical assistance, Mark Richards for t h e ceruloplasmin assays, and G a r y Peck for assistance in preparing t h e figures. Special t h a n k s is e x t e n d e d t o Allen Edgar for t h e original isolate of t h e Eimeria species.
REFERENCES
FIG. 2. Electrophoretic separation of plasma lipoproteins of control (A), E. meleagrimitis-infected (B), and E. adenoeides-infected (C) 3-week-old turkeys. Four fractions are shown: portomicrons (PM), /?-, pre-0 (p/3)-, and a-lipoproteins. Arrow indicates point of application of plasma.
Therefore, hemolysis t h a t often occurs as severely-infected p o u l t s are bled did n o t interfere with t h e separation or q u a n t i t a t i o n of serum p r o t e i n fractions. E l e c t r o p h o r e t i c separation and staining with Oil R e d 0 showed four lipoprotein fractions: p o r t o m i c r o n s , /}-, pre-/?-, and a-lipoproteins (Fig. 2). Because t h e poults were n o t fasted before blood samples were t a k e n , p o r t o m i c r o n s were present in b o t h controls and infected plasma, b u t t h e y were m o r e c o n c e n t r a t e d in t h e controls. Beta and a-lipoproteins were identi-
Augustine, P. C , 1982. Effect of feed and water deprivation on organ and blood characteristics of young turkeys. Poultry Sci. 61:796-799. Augustine, P. C , and O. P. Thomas, 1979. Eimeria meleagrimitis in young turkeys: effects on weight, blood, and organ parameters. Avian Dis. 24:854-862. Augustine, P. C , and O. P. Thomas, 1981. Effect of time on response to Eimeria adenoeides and Eimeria meleagrimitis infection in young turkeys. Avian Dis. 25:366-373. Gelman Instrument Company, 1978. Lipoprotein electrophoresis system. Tech. Bull. 28. Kelsey, R. L., 1969. Interpretation of routine electrophoretic patterns of serum proteins in health and disease. In Gelman Manual of Clinical Electrophoresis. Ann Arbor, MI. Lynch, J. E., and H. J. Stafseth, 1953. Electrophoretic studies on the serum proteins of turkeys. The composition of normal turkey serum. Poultry Sci. 32:1068-1073. Ruff, M. D., and P. C. Augustine, 1982. Effects of coccidiosis on the electrophoretic pattern of serum proteins in chickens. J. Parasitol. 68:107— 111. Smith, B.S.W., and H. Wright, 1974. Improved manual and automated procedures for estimation of ceruloplasmin oxidase activity. Clin. Chim. Acta 50:359-366. Sturkie, P. D., 1976. Avian Physiology. P. D. Sturkie, ed. Springer-Verlag, New York, NY.
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ACKNOWLEDGMENTS