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Hematological Response of Immunized and Serially Bled Japanese Quail1,2
Hematological Response of Immunized and Serially Bled Japanese Quail1'2 R. P. GILDERSLEEVE and J. P. THAXTON Department of Poultry Science, North Carolina State University, Raleigh, North Carolina 27695-7608 M. J. GALVIN and D. I. McREE Laboratory of Behavioral and Neurological Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 (Received for publication December 27, 1984)
1985 Poultry Science 64:1806-1808
INTRODUCTION T h e i m m u n o b i o l o g y of J a p a n e s e quail has been studied (Leslie and Benedict, 1 9 6 9 ; Aitken and Parry, 1 9 7 4 ; Benton et al, 1 9 7 7 ; Lasley, 1 9 8 0 ) , and this bird is accepted as a m o d e l for t h e s t u d y of microwave exposure in ovo o n immunological a n d hematological variables (Hamrick et al., 1 9 7 7 ; Galvin et al, 1 9 8 1 ) . T h e conventional m e t h o d s of assessing all h u m o r a l and s o m e cell m e d i a t e d i m m u n e responses in birds (Glick, 1 9 7 7 ) n o r m a l l y involve repetitive blood sampling, i.e., serial bleedings. T h e q u e s t i o n of w h e t h e r serial bleedings, which are believed t o cause transient anemia followed b y hematological compensatory responses in J a p a n e s e quail and o t h e r birds (Natt and Herrick, 1 9 5 5 ; Gildersleeve et al, 1985), influence i m m u n e responsiveness has n o t been answered. Therefore, t h e objective of this s t u d y was t o d e t e r m i n e if serial bleedings,
'Paper No. 9650 of the Journal Series of the North Carolina Agricultural Research Service, North Carolina State University, Raleigh, NC 27695-7601. 2 The use of trade names in this publication does not constitute endorsement by the North Carolina Agricultural Research Service of the product mentioned nor criticism of similar ones not mentioned.
which cause compensatory hematological responses in Japanese quail, would alter t h e b i r d ' s ability t o m o u n t a p r i m a r y h u m o r a l i m m u n e response. MATERIALS AND METHODS T h e A t h e n s R a n d o m b r e d J a p a n e s e quail were m a i n t a i n e d in individual cages o n a 16-hr p h o t o p e r i o d at 2 5 ± 2 C. Quail layer feed (24% protein, 2 8 1 1 kcal metabolizable energy (ME)/kg, 2 . 5 5 % Ca) a n d w a t e r were provided ad libitum. Twelve-week-old sexually m a t u r e female J a p a n e s e quail were i m m u n i z e d b y intravenous injection w i t h .5 m l of a 7.5% ( a p p r o x i m a t e l y 1 X 1 0 7 cells) suspension of Chukar partridge (Alectoris graeca chukar) red b l o o d cells (CRBC; B e n t o n et al, 1977). Blood samples (1 m l f r o m t h e jugular vein) were collected either serially or once from separate g r o u p s of quail at 0 ( n o n i m m u n i z e d ) , 3, 6, a n d 9 days p o s t i m m u n i z a t i o n . Serial bleedings m e a n t h a t each quail was bled (1 ml) at each designated t i m e post-CRBC immunization, and single bleedings m e a n t h a t a quail was bled (1 ml) only o n c e ; i.e., at t h e designated t i m e postCRBC i m m u n i z a t i o n . T h e b o d y weight of all quail was 130 ± 2 g ( m e a n ± SE). O n e milliliter of b l o o d loss
1806
Downloaded from http://ps.oxfordjournals.org/ at East Tennessee State University on June 18, 2015
ABSTRACT Sexually mature female Japanese quail (Coturnix coturnix japonica) were immunized by an intravenous injection with .5 ml of a 7.5% suspension of Chukar partridge (Alectoris graeca chukar) red blood cells (CRBC). Blood samples were collected either serially from one group of quail or a single time from separate groups of quail at 0 (nonimmunized), 3, 6, and 9 days postimmunization. Total anti-CRBC hemagglutinin titers (HA) were measured by a microhemagglutinin procedure. Selected hematological variables were also measured. Mean HA was not affected by serial blood sampling. Total erythrocyte numbers, percent hematocrit, and hemoglobin levels were depressed in serially bled quail at 3 days postimmunization. Reticulocytosis was found in serially bled quail at 6 days postimmunization. (Key words: Japanese quail, hemagglutinin, hematocrit, reticulocyte, anemia)
RESEARCH NOTE
DAYS POST-CRBC
FIG. 1. Effect of single (open bars) and serial (closed bars) blood collections on mean + SE (10 < n < 15) reticulocytes, hemoglobin, total erythrocyte numbers (TRBC), and hematocrits in Chukar partridge red blood cells (CRBC) immunized, sexually mature female Japanese quail (*P<.05, by t test).
represented about 11% of the total blood volume (Nirmalan and Robinson, 1972). Total anti-CRBC hemagglutinins (HA) were determined in serum by a microhemagglutinin procedure (Witlin, 1967; Benton et al, 1977). A Hemolab Electrozone Celloscope (Particle Data, Inc., Elmhurst, IL) was used to determine total erythrocyte number (TRBC) and hematocrit (Hct). Hemoglobin levels (Hb; g/dl) were determined by the cyanomethoglobin method of Sunderman et al. (1953). The percentage of reticulocytes were determined from whole blood smears after supravital staining with new methylene blue (Lucas and Jamroz, 1961). Statistical analyses were performed with the Statistical Analysis System (SAS, 1982) using paired t tests. Percentages were subjected to arc sine transformations and anti-CRBC HA titers were converted to log2 values prior to analysis. Statements of significance are based on a probability of .05.
RESULTS AND DISCUSSION The hematological profile of quail bled serially and singly is depicted in Figure 1. These data agree with those of a previous report from this laboratory (Gildersleeve et al, 1985) that blood loss in Japanese quail causes a transient anemia followed by hematological compensation. The hematological profile was characterized by significant decreases in TRBC, HCT, and Hb after two of four serial bleedings. Additionally, the hematological compensation to the anemia was evidenced by a significant increase in reticulocytes after the third serial bleeding. These data suggest that increased erythropoiesis by the bone marrow is integral to hematological compensation in Japanese quail. As shown in Figure 2, the primary humoral immune response to CRBC was not affected by the serial bleedings. In fact, the immune response of the serially-bled quail paralleled that of the singly-bled quail in time and magnitude of maximum HA levels. Furthermore,
Downloaded from http://ps.oxfordjournals.org/ at East Tennessee State University on June 18, 2015
DAYS POST- CRBC
1807
1808
GILDERSLEEVE ET AL. loss. However, this is an intriguing q u e s t i o n and it merits additional e x p e r i m e n t a t i o n .
10 r
ACKNOWLEDGMENTS T h e a u t h o r s t h a n k P. V. Phelps, R. P. Wiard, D. Sugg, G. S. Swann, R. L. Hunter, and R. M. Mitchell for their excellent assistance during t h e course of this study. This s t u d y was s u p p o r t e d in part b y a National Institute of Environmental Health Sciences contract NOl-ES-2-5109.
REFERENCES
POST-CRBC
FIG. 2. Mean ± SE (10 < n < 15) anti-Chukar partridge red blood cells (CRBC) hemagglutinin titer in sexually mature female Japanese quail after single (—•—) and serial (—*—) blood collections. t h e early part of t h e degradation phase of t h e response, as indicated b y anti-CRBC levels at 9 days post-CRBC challenge, was n o t different between t h e two treatment groups. In previous studies, Gildersleeve et al. ( 1 9 8 5 ) d e m o n s t r a t e d t h a t b l o o d loss, which was comparable t o t h a t in t h e present s t u d y , resulted in a transient r e d u c t i o n in t o t a l leukocyte n u m b e r s . However, t h e changes in relative percentages and n u m b e r s of each l e u k o c y t e cell t y p e were m o r e c o m p l e x . These a u t h o r s also r e p o r t e d t h a t l y m p h o c y t e n u m b e r s in n o n immunized, b u t p h l e b o t o m i z e d , quail decreased initially a n d r e t u r n e d t o basal levels a t 3 days p o s t p h l e b o t o m y . C o m p e n s a t o r y changes in n u m b e r s of t h e o t h e r l e u k o c y t i c t y p e s w e r e n o t as apparent. T h e objective of this s t u d y was t o determine if hematological c o m p e n s a t i o n t o multiple bleedings w o u l d affect t h e ability of quail t o express a n o r m a l p r i m a r y h u m o r a l i m m u n e response. T h e i n t e n t of this s t u d y was n o t t o assess t h e alternate question of w h e t h e r t h e expression of a h u m o r a l i m m u n e response w o u l d influence hematological responsiveness t o blood
Downloaded from http://ps.oxfordjournals.org/ at East Tennessee State University on June 18, 2015
DAYS
Aitken, I. D., and S. H. Parry, 1974. Comparative serological response of the chicken, pheasant and quail to a soluble and particulate antigen. Immunol. 27:623-629. Benton, E. H., G. W. Morgan, P. Thaxton, C. R. Parkhurst, and M. O. Shambley, 1977. Antibody responses to xenogenic red blood cell challenge in the Japanese quail. Immunol. Commun. 6: 259-265. Galvin, M. J., D. I. McRee, C. A. Hall, J. P. Thaxton, and C. R. Parkhurst, 1981. Humoral and cellmediated immune function in adult Japanese quail following exposure to 2.45 GHz microwave radiation during embryogeny. Bioelectromagnetics 2:269-278. Gildersleeve, R. P., M. J. Galvin, J. P. Thaxton, and D. I. McRee, 1985. Hematological response of Japanese quail to acute hemorrhagic stress. Comp. Biochem. Physiol. 81A:403-409. Glick, B., 1977. The bursa of Fabricius and immunoglobulin synthesis. Int. Rev. Cytol. 48:345—402. Hamrick, P. E., D. I. McRee, P. Thaxton, and C. R. Parkhust, 1977. Humoral immunity of Japanese quail subjected to microwave radiation during embryogeny. Health Phys. 33:23-33. Lasley, J. A., 1980. An assessment of cell-mediated immune responses in Japanese quail. M.S. Thesis, North Carolina State University, Raleigh, NC. Leslie, G. A., and A. A. Benedict, 1969. Structural and antigenic relationships between avian immunoglobulins. 1. The immune responses of pheasants and quail and reductive dissociation of their immunoglobulins. J. Immunol. 103:1356—1365. Lucas, A. M., and C. Jamroz, 1961. Pages 2 3 0 - 2 3 1 in Atlas of Avian Hematology, U.S. Dept. Agric, Washington, DC. Natt, M. P., and C. A. Herrick, 1955. The effect of cecal coccidiosis on the blood cells of domestic fowl. Poultry Sci. 34:1100-1106. Nirmalan, G. P., and G. A. Robinson, 1972. Effect of age, sex and egg laying on the total erythrocyte volume ( s l CrO„ label) and the plasma volume ('"i-serum albumin label) of Japanese quail. Can. J. Physiol. Pharmacol. 50:6-10. SAS, 1982. SAS User's Guide: Statistics. SAS Inst., Inc., Cary, NC. Sunderman, F. W., R. P. MacFate, D. A. MacFadyen, G. F. Stevenson, and B. E. Copeland, 1953. Symposium on clinical hemoglobinometry. Am. J. Clin. Pathol. 23:519-598. Witlin, B., 1967. Detection of antibodies by microtitration techniques. Mycopathol. Mycol. Appl. 33:241-252.
1985 Poultry Science 64:1806-1808
INTRODUCTION T h e i m m u n o b i o l o g y of J a p a n e s e quail has been studied (Leslie and Benedict, 1 9 6 9 ; Aitken and Parry, 1 9 7 4 ; Benton et al, 1 9 7 7 ; Lasley, 1 9 8 0 ) , and this bird is accepted as a m o d e l for t h e s t u d y of microwave exposure in ovo o n immunological a n d hematological variables (Hamrick et al., 1 9 7 7 ; Galvin et al, 1 9 8 1 ) . T h e conventional m e t h o d s of assessing all h u m o r a l and s o m e cell m e d i a t e d i m m u n e responses in birds (Glick, 1 9 7 7 ) n o r m a l l y involve repetitive blood sampling, i.e., serial bleedings. T h e q u e s t i o n of w h e t h e r serial bleedings, which are believed t o cause transient anemia followed b y hematological compensatory responses in J a p a n e s e quail and o t h e r birds (Natt and Herrick, 1 9 5 5 ; Gildersleeve et al, 1985), influence i m m u n e responsiveness has n o t been answered. Therefore, t h e objective of this s t u d y was t o d e t e r m i n e if serial bleedings,
'Paper No. 9650 of the Journal Series of the North Carolina Agricultural Research Service, North Carolina State University, Raleigh, NC 27695-7601. 2 The use of trade names in this publication does not constitute endorsement by the North Carolina Agricultural Research Service of the product mentioned nor criticism of similar ones not mentioned.
which cause compensatory hematological responses in Japanese quail, would alter t h e b i r d ' s ability t o m o u n t a p r i m a r y h u m o r a l i m m u n e response. MATERIALS AND METHODS T h e A t h e n s R a n d o m b r e d J a p a n e s e quail were m a i n t a i n e d in individual cages o n a 16-hr p h o t o p e r i o d at 2 5 ± 2 C. Quail layer feed (24% protein, 2 8 1 1 kcal metabolizable energy (ME)/kg, 2 . 5 5 % Ca) a n d w a t e r were provided ad libitum. Twelve-week-old sexually m a t u r e female J a p a n e s e quail were i m m u n i z e d b y intravenous injection w i t h .5 m l of a 7.5% ( a p p r o x i m a t e l y 1 X 1 0 7 cells) suspension of Chukar partridge (Alectoris graeca chukar) red b l o o d cells (CRBC; B e n t o n et al, 1977). Blood samples (1 m l f r o m t h e jugular vein) were collected either serially or once from separate g r o u p s of quail at 0 ( n o n i m m u n i z e d ) , 3, 6, a n d 9 days p o s t i m m u n i z a t i o n . Serial bleedings m e a n t h a t each quail was bled (1 ml) at each designated t i m e post-CRBC immunization, and single bleedings m e a n t h a t a quail was bled (1 ml) only o n c e ; i.e., at t h e designated t i m e postCRBC i m m u n i z a t i o n . T h e b o d y weight of all quail was 130 ± 2 g ( m e a n ± SE). O n e milliliter of b l o o d loss
1806
Downloaded from http://ps.oxfordjournals.org/ at East Tennessee State University on June 18, 2015
ABSTRACT Sexually mature female Japanese quail (Coturnix coturnix japonica) were immunized by an intravenous injection with .5 ml of a 7.5% suspension of Chukar partridge (Alectoris graeca chukar) red blood cells (CRBC). Blood samples were collected either serially from one group of quail or a single time from separate groups of quail at 0 (nonimmunized), 3, 6, and 9 days postimmunization. Total anti-CRBC hemagglutinin titers (HA) were measured by a microhemagglutinin procedure. Selected hematological variables were also measured. Mean HA was not affected by serial blood sampling. Total erythrocyte numbers, percent hematocrit, and hemoglobin levels were depressed in serially bled quail at 3 days postimmunization. Reticulocytosis was found in serially bled quail at 6 days postimmunization. (Key words: Japanese quail, hemagglutinin, hematocrit, reticulocyte, anemia)
RESEARCH NOTE
DAYS POST-CRBC
FIG. 1. Effect of single (open bars) and serial (closed bars) blood collections on mean + SE (10 < n < 15) reticulocytes, hemoglobin, total erythrocyte numbers (TRBC), and hematocrits in Chukar partridge red blood cells (CRBC) immunized, sexually mature female Japanese quail (*P<.05, by t test).
represented about 11% of the total blood volume (Nirmalan and Robinson, 1972). Total anti-CRBC hemagglutinins (HA) were determined in serum by a microhemagglutinin procedure (Witlin, 1967; Benton et al, 1977). A Hemolab Electrozone Celloscope (Particle Data, Inc., Elmhurst, IL) was used to determine total erythrocyte number (TRBC) and hematocrit (Hct). Hemoglobin levels (Hb; g/dl) were determined by the cyanomethoglobin method of Sunderman et al. (1953). The percentage of reticulocytes were determined from whole blood smears after supravital staining with new methylene blue (Lucas and Jamroz, 1961). Statistical analyses were performed with the Statistical Analysis System (SAS, 1982) using paired t tests. Percentages were subjected to arc sine transformations and anti-CRBC HA titers were converted to log2 values prior to analysis. Statements of significance are based on a probability of .05.
RESULTS AND DISCUSSION The hematological profile of quail bled serially and singly is depicted in Figure 1. These data agree with those of a previous report from this laboratory (Gildersleeve et al, 1985) that blood loss in Japanese quail causes a transient anemia followed by hematological compensation. The hematological profile was characterized by significant decreases in TRBC, HCT, and Hb after two of four serial bleedings. Additionally, the hematological compensation to the anemia was evidenced by a significant increase in reticulocytes after the third serial bleeding. These data suggest that increased erythropoiesis by the bone marrow is integral to hematological compensation in Japanese quail. As shown in Figure 2, the primary humoral immune response to CRBC was not affected by the serial bleedings. In fact, the immune response of the serially-bled quail paralleled that of the singly-bled quail in time and magnitude of maximum HA levels. Furthermore,
Downloaded from http://ps.oxfordjournals.org/ at East Tennessee State University on June 18, 2015
DAYS POST- CRBC
1807
1808
GILDERSLEEVE ET AL. loss. However, this is an intriguing q u e s t i o n and it merits additional e x p e r i m e n t a t i o n .
10 r
ACKNOWLEDGMENTS T h e a u t h o r s t h a n k P. V. Phelps, R. P. Wiard, D. Sugg, G. S. Swann, R. L. Hunter, and R. M. Mitchell for their excellent assistance during t h e course of this study. This s t u d y was s u p p o r t e d in part b y a National Institute of Environmental Health Sciences contract NOl-ES-2-5109.
REFERENCES
POST-CRBC
FIG. 2. Mean ± SE (10 < n < 15) anti-Chukar partridge red blood cells (CRBC) hemagglutinin titer in sexually mature female Japanese quail after single (—•—) and serial (—*—) blood collections. t h e early part of t h e degradation phase of t h e response, as indicated b y anti-CRBC levels at 9 days post-CRBC challenge, was n o t different between t h e two treatment groups. In previous studies, Gildersleeve et al. ( 1 9 8 5 ) d e m o n s t r a t e d t h a t b l o o d loss, which was comparable t o t h a t in t h e present s t u d y , resulted in a transient r e d u c t i o n in t o t a l leukocyte n u m b e r s . However, t h e changes in relative percentages and n u m b e r s of each l e u k o c y t e cell t y p e were m o r e c o m p l e x . These a u t h o r s also r e p o r t e d t h a t l y m p h o c y t e n u m b e r s in n o n immunized, b u t p h l e b o t o m i z e d , quail decreased initially a n d r e t u r n e d t o basal levels a t 3 days p o s t p h l e b o t o m y . C o m p e n s a t o r y changes in n u m b e r s of t h e o t h e r l e u k o c y t i c t y p e s w e r e n o t as apparent. T h e objective of this s t u d y was t o determine if hematological c o m p e n s a t i o n t o multiple bleedings w o u l d affect t h e ability of quail t o express a n o r m a l p r i m a r y h u m o r a l i m m u n e response. T h e i n t e n t of this s t u d y was n o t t o assess t h e alternate question of w h e t h e r t h e expression of a h u m o r a l i m m u n e response w o u l d influence hematological responsiveness t o blood
Downloaded from http://ps.oxfordjournals.org/ at East Tennessee State University on June 18, 2015
DAYS
Aitken, I. D., and S. H. Parry, 1974. Comparative serological response of the chicken, pheasant and quail to a soluble and particulate antigen. Immunol. 27:623-629. Benton, E. H., G. W. Morgan, P. Thaxton, C. R. Parkhurst, and M. O. Shambley, 1977. Antibody responses to xenogenic red blood cell challenge in the Japanese quail. Immunol. Commun. 6: 259-265. Galvin, M. J., D. I. McRee, C. A. Hall, J. P. Thaxton, and C. R. Parkhurst, 1981. Humoral and cellmediated immune function in adult Japanese quail following exposure to 2.45 GHz microwave radiation during embryogeny. Bioelectromagnetics 2:269-278. Gildersleeve, R. P., M. J. Galvin, J. P. Thaxton, and D. I. McRee, 1985. Hematological response of Japanese quail to acute hemorrhagic stress. Comp. Biochem. Physiol. 81A:403-409. Glick, B., 1977. The bursa of Fabricius and immunoglobulin synthesis. Int. Rev. Cytol. 48:345—402. Hamrick, P. E., D. I. McRee, P. Thaxton, and C. R. Parkhust, 1977. Humoral immunity of Japanese quail subjected to microwave radiation during embryogeny. Health Phys. 33:23-33. Lasley, J. A., 1980. An assessment of cell-mediated immune responses in Japanese quail. M.S. Thesis, North Carolina State University, Raleigh, NC. Leslie, G. A., and A. A. Benedict, 1969. Structural and antigenic relationships between avian immunoglobulins. 1. The immune responses of pheasants and quail and reductive dissociation of their immunoglobulins. J. Immunol. 103:1356—1365. Lucas, A. M., and C. Jamroz, 1961. Pages 2 3 0 - 2 3 1 in Atlas of Avian Hematology, U.S. Dept. Agric, Washington, DC. Natt, M. P., and C. A. Herrick, 1955. The effect of cecal coccidiosis on the blood cells of domestic fowl. Poultry Sci. 34:1100-1106. Nirmalan, G. P., and G. A. Robinson, 1972. Effect of age, sex and egg laying on the total erythrocyte volume ( s l CrO„ label) and the plasma volume ('"i-serum albumin label) of Japanese quail. Can. J. Physiol. Pharmacol. 50:6-10. SAS, 1982. SAS User's Guide: Statistics. SAS Inst., Inc., Cary, NC. Sunderman, F. W., R. P. MacFate, D. A. MacFadyen, G. F. Stevenson, and B. E. Copeland, 1953. Symposium on clinical hemoglobinometry. Am. J. Clin. Pathol. 23:519-598. Witlin, B., 1967. Detection of antibodies by microtitration techniques. Mycopathol. Mycol. Appl. 33:241-252.