SZ. Li*,
K. E. Nestor1*,
Y. M. Saif†,
W. L. Bacon*,
and J. W. Anderson*
*The Ohio State University Department of Animal Sciences
†Ohio Agricultural Research and Development Center Food Animal Health Research Program
1 For more information, contact at: The Ohio State University,
Ohio Agricultural Research and Development Center, 125 Gerlaugh Hall,
1680 Madison Ave., Wooster, OH 44691; 330-263-3757; Fax: 330-263-3949;
e-mail: nestor.1@osu.edu.
Mitogenic responses were examined for purified peripheral blood mononuclear cells (PBMC) and whole blood from individuals in a line (F) of turkeys selected for increased 16-week body weight and its corresponding randombred control (RBC2). The PBMC were isolated by centrifugation over Histopaque-1077 density gradient and tested for mitogenic responses to concanavalin A (ConA; 25 µg/mL) and phytohemagglutinin M (PHA-M; 100 µg/mL). For the whole blood assay, six-week-old poults from both lines were injected with inactivated Pasteurella multocida. Heparinized blood samples were collected prior to injection (0 days) and at 2, 4, 7, and 14 days postinjection. The diluted whole blood was then tested for the mitogenic responses to Con A (25 µg/mL) and PHA-M (25 µg/mL). The cultures were then pulsed with 3H-thymidine, and incorporation was measured using a liquid scintillation counter.
There was a line difference in the mitogenic responses to ConA for PBMC and whole blood assays, but no line difference was observed in the response to PHA-M for both assays. For the purified PBMC assay, the F line had a lower response than its randombred control line (P <= 0.05) to Con A expressed as either cpm or a stimulation index (SI; ratio of cpm for stimulated cells to the cpm for unstimulated cells). For the whole blood assay, the F line had generally lower SI values in the responses to Con A than the RBC2 line with differences being significant at 0 and 2 days postinjection (P <= 0.01) and at 14 days postinjection (P <= 0.05). Genetic selection for increased body weight may have affected the lymphoblastogenic potential of Line F that could affect disease resistance.
Host resistance to pathogens is complicated and involves both specific and nonspecific resistant factors. The humoral immune response is the principle specific immunity against extracellular bacteria, whereas the cell-mediated immunity plays a major role in the responses against intracellular bacteria and viruses (Abbas et al., 1994). Different genetic backgrounds in individuals may vary in these facets of host immunity and influence resistance to infectious diseases.
Previous reports showed that a line (F) of turkeys selected for increased 16-week body weight was more susceptible to erysipelas, fowl cholera, and Newcastle disease than its parental randombred control line (RBC2) (Saif et al., 1984; Sacco et al., 1991; Tsai et al., 1992; Nestor et al., 1996b). However, the antibody titers detected with a hemagglutination inhibition test were higher in the surviving F-line turkeys than those of the RBC2 line in a challenge experiment with Newcastle disease virus (Tsai et al., 1992). In addition, the F line had higher antibody responses to Newcastle disease virus vaccines as measured by ELISA (Sacco et al., 1994). These results suggest that there was no positive correlation between resistance to a specific disease and humoral response in these turkeys. Therefore, it is appropriate to suggest that other immune mechanisms, such as cell-mediated immunity, may be contributing to the explanation of the susceptibility of the F line. The mitogenic response assay is a practical measure of cell-mediated immunity. The purpose of the present study was to examine the mitogenic responses to plant lectins in the F and RBC2 lines of turkeys.
Six-week-old poults from each line were used in the experiments. The RBC2 line was a randombred control line initiated in 1966 (Nestor, 1977a). The F line was a subline of RBC2 selected for increased 16-week body weight (Nestor, 1977b, 1984; Nestor et al., 1996a). The inbreeding coefficient of the RBC2 and F lines was 12.6 and 27.1%, respectively, at the time of the study. The birds were provided feed (Naber and Touchburn, 1970) and water for ad libitum consumption. The lines were grown intermingled in each experiment.
For the mitogenic responses of purified peripheral blood mononuclear cells (PBMC), 2 mL of heparinized blood were collected from 10 RBC2 and 14 F line individuals of equal numbers of males and females. The PBMC were isolated by centrifugation over Histopaque-1077 (Sigma Chemical Co., St. Louis, MO 63178-9916) density gradient, and cell viability was assessed by the means of trypan blue dye exclusion. Cells were then enumerated and adjusted to 1 x 107 cell /mL in RPMI 1640 medium (Sigma Chemical Co., St. Louis, MO 63178-9916) containing penicillin (100 units/mL), streptomycin (100 µg/mL), and 5% complement-inactivated (56ºC for 30 min) chicken serum (Sigma Chemical Co., St. Louis, MO 63178-9916). Cells were cultured at a concentration of 1 x 106 cells in 200 uL/well in 96-well culture plates under stimulation in triplicate in vitro with either concanavalin A (Con A; Sigma Chemical Co., St. Louis, MO 63178-9916) (25 µg/mL), or phytohemagglutinin-M (PHA-M; Boehringer Mannheim Corporation, Indianapolis, IN 46250-0414) (100 µg/mL), or with medium alone. Cells were incubated for 52 hours at 39ºC in a humidified incubator with 5% CO2, then pulsed with 20 µL of 3H-thymidine (1 µCi, 6.7 Ci/mmol) for 16 hours. Cells were harvested on glass fiber filters by using a cell harvester (Skatron, Inc., Sterline, VA 20166). The 3H-thymidine incorporation was measured using a liquid scintillation counter.
A whole blood assay was carried out according to the methods described by Sharma and Belzer (1992), with some modification. Briefly, 14 turkeys from each line were subcutaneously injected with 1 mL of formaldehyde-inactivated Pasteurella multocida (1.2 x 107 bacteria/mL) at six weeks of age. Heparinized blood samples were collected before injection (0 days) and at 2, 4, 7, and 14 days postinjection. Whole blood cells were diluted 1:20 with RPMI-1640 containing penicillin (100 units/mL), streptomycin (100 µg/mL), 5 x 10-5M 2-mercaptoethanol, and 2% pooled and heat-inactivated (56ºC for 30 min) turkey serum. One hundred microliters of diluted blood was dispensed into the 96-well flat-bottomed culture plates which contained 100 µL of Con A (50 ug/mL), or PHA-M (50 ug/mL), or medium alone in triplicate. Cultures were grown at 40ºC for 52 hours in 5% CO2 humidified incubator. The cell cultures were then labeled with 20 µL of 3H-thymidine (1 µCi ) for an additional 16 hours. Cells were lysed by repeated freezing (at -20ºC) and thawing three times and were harvested for liquid scintillation counting as noted previously.
Means of counts per minute were calculated for triplicate cultures. Results for each PBMC sample were expressed as the mean counts per minute for cells stimulated by ConA or PHA-M minus the mean counts per minute of background. A stimulation index (SI) was obtained by dividing the average counts per minute from stimulated cells by the average counts per minute from unstimulated cells. An ANOVA was used to test for the significant differences in the mitogenic responses between lines and sexes using the General Linear Model procedure of SAS® (SAS Institute, 1988). All data were expressed as the least squares mean ± SEM.
There was no significant sex effect on mitogenic responses for either purified PBMC or whole blood. However, there was a significant line effect for Con A but not for PHA-M stimulation of PBMC (Table 1). The RBC2 line had significantly higher responses to Con A when the results were expressed either as counts per minute or SI (P <= 0.05).
For the mitogenic responses tested with the whole blood assay, the results showed that mitogenic responses to Con A expressed as SI were greater in the RBC2 line than in the F line with differences being significant at 0 and 2 days postinjection (both P <= 0.01) and at 14 days postinjection (P <= 0.05). The SI values from both lines peaked at 2 days postinjection. However, there was no significant difference in the responses to PHA-M between the two lines.
| Table 1. Mitogenic Responses to Concanavalin (Con A) and Phytohemagglutinin-M (PHA-M) of Peripheral Blood Mononuclear Cells from the RBC2 and F Lines of Turkeys (Least Squares Mean ± SEM). | ||
|---|---|---|
| Line1 | ||
| Mitogen | RBC2 | F |
| Counts per minute x 10-3 | ||
| Con A | 23.9 ± 3.5a | 10.9 ± 2.9b |
| PHA-M | 9.9 ± 2.7 | 14.4 ± 2.2 |
| Stimulation Index(SI)2 | ||
| Con A | 29.6 ± 5.8a | 8.3 ± 4.9b |
| PHA-M | 14.4 ± 3.7 | 11.8 ± 3.1 |
| a,b Means within a row with no common superscript differ significantly (P <= 0.05). 1 RBC2= a randombred control line and F = a subline of the RBC2 line selected for increased 16-week body weight. The number of samples was 10 and 14 for Lines RBC2 and F, respectively. 2 SI = ratio of the cpm for stimulated cultures to the cpmfor unstimulated cultures. | ||
The mitogenic response to plant lectins is conventionally used to measure the cell-mediated immunity in mammals and aves. The Con A and PHA mitogens stimulate T lymphocytes (Toivanen and Toivanen, 1973; Hovi et al., 1978) by indirectly cross-linking the T cell receptor complex (Abbas et al., 1994). Compared with the assays using purified PBMC, whole blood assays are easy, rapid, more valid, and a true measure of cellular immune competence, and therefore suitable for monitoring the functional capabilities of immune cells in avian flocks (Lee, 1978; Sharma and Belzer, 1992; Talebi et al., 1995). However, there is individual variation in the whole blood assay (Sharma and Belzeer, 1992) so both the purified PMBC and whole blood assays were used in the present study.
In separate studies, the counts per minute and SI of purified PBMC were lower than those of cells prepared from buffy coat possibly because the preparation of the lymphocytes through Histopaque-1077 caused lower responses to mitogens (unpublished data). It has been reported that Ficoll (sodium diatrizoate is a major component and also is the main component of Histopaque-1077) may have some inhibitory effect on the mitogenic stimulation of lymphocytes (Lee, 1974; Maheswaran et al., 1975). In addition, the ratio of cell types may vary with different procedures of cell preparation. Erf and Smyth (1996) reported that PBMC prepared through Histopaque-1077 may contain more thrombocytes and monocytes in addition to lymphocytes, whereas cells obtained from buffy coats by slow-speed centrifugation may be rich in lymphocytes and some contamination with erythrocytes and thrombocytes, but contain no monocytes. However, there was less variation among individuals within lines in the mitogenic responses with purified PBMC than with buffy coat cells (unpublished data). The mitogenic responses are also affected by many other parameters, such as the storage time (Raj et al., 1997), the enhancement by erythrocytes in the cultures (Powell, 1980), the suppression by monocytes (Vainio and Ratcliffe, 1984; Schaefer et al., 1985), serum sources and concentrations, incubation temperature, and length of assay (Lee, 1974, 1978; Maheswaran et al., 1975; Sharma and Belzer, 1992; Talebi et al., 1995).
The results herein demonstrated that the F line had lower responses to Con A using either purified PBMC or whole blood than the RBC2 line, but there was no line difference in the responses to PHA-M. In the subsequent challenge experiment with virulent P. multocida, it was observed that the poults that died earlier in the F line usually had smaller SI values for Con A-stimulated PBMC (data not shown). The Pearson correlation coefficient was 0.40 (P = 0.052) between SI for Con A and the number of days to death post-injection with P. multocida. The correlation coefficient between the SI for PHA and days to death was not significant. The mortality following challenge with P. multocida was 30 and 85.7% for the RBC2 and F line, respectively.
The results indicated that mitogenic responses to Con A may be related to resistance to specific diseases. The different responses to Con A in the two lines of turkeys may be due to genetic variation. In the present study, there was no significant statistical difference in mitogenic responses to PHA-M between two lines. Different gene systems may mediate blood lymphocyte responsiveness to Con A and PHA in chickens (Miggiano et al., 1976; Morrow and Abplanalp, 1981; Fredericksen and Silmour, 1983). In addition, the mitogenic response to Con A is suggested to be controlled by at least two genes - a non-major histocompatibility complex-associated gene (ConA1) and a major histocompatibility complex-associated gene (ConA2) (Morrow and Abplanalp, 1981; Knudtson et al., 1990).
Previous results have shown that the F line was susceptible to several infectious diseases. It was not possible to correlate the susceptibility of the F line to Newcastle disease with antibody response (Tsai et al., 1992; Sacco et al., 1994), or with known changes in the frequency of the MHC Class II haplotypes for both P. multocida and Newcastle disease virus (Nestor et al., 1996c). In highly inbred chicken lines, Knudtson et al. (1990) found that the level of inteleukin-2-like activity was associated with the level of mitogenic response to Con A. Selection for increased body weight in the F line may have resulted in changes of gene frequency for genes controlling mitogenic responses to Con A, cytokines, and other facets of cell-mediated immunity, therefore affecting disease resistance. The mitogenic response to Con A in whole blood assay may have a potential for use as an indicator for genetic selection for improved disease resistance.
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