June 2008 Journal of northeast Agricultural University ol.15No.226-29 Effects of Immunopotentiator of the Traditional Chinese Medicine on T Lymphocytes in Chicken Blood SHI Qiumei, LI Chunling GAo Guisheng, SHEN Ping, and TANG Shengling 2 Veterinary Research Institute, Guangdong Provincial Academy of Agricultural Sciences, Guangzhou 510640,China Abstract: In order to investigate the mechanism of action of immunoenhancer, the effects of the traditional Chinese medicine oter on the quantity and the transformation rates of T lymphocytes in the chicken blood were determined. Total 120 chickens were randomly assigned into three groups. The 1% and the 0.5% of the Chinese medicine immunopromoter were added to the chicken drinking water, respectively. The quantity of T lymphocytes in each group was measured by a-Naphthyl acetate esterase(ANAE)staining. The results showed that the percentages of T lymphocytes of the treatment groups were significantly higher than that of the control group(P<0.05), and the percentage of the 1% group significantly higher than that of the 0.5% group (P<0.05). In conclusion, the transformation rates of T lymphocytes showed that the Chinese medicine immunopromoter had the significant enhancing effect on the transformation rates of T lymphocytes of the treated chickens. The traditional Chinese medicine amunopromoter had the distinct function to promote the quantity and the transformation rate of T lymphocytes Key words: traditional Chinese medicine immunopromoter, T lymphocyte, a-Naphthyl acetate esterase staining CLC number: S831; S852 16 Document code: A Article ID: 1006-8104(2008)-02-0026-04 phages so that the body can obtain the high-level Introduction immune response after vaccination and strengthen the resistance to diseases. The immune function of the There are still outbreaks of infectious diseases in animal directly affects its resistance to diseases and vaccinated chickens and the gradually increasing the immune effect after vaccination. The level of the dosage fails to achieve the ideal therapeutic effects. immune function of the animal is closely related to The reason may be the decreasing immune function the quantity of T and B lymphocytes. It is generally of the body due to the long-term mindless use of accepted that the function of T lymphocytes can pro- medicine and unreasonable immune vaccination, duce lymphokines to increase the immunity and specially when the development of lymphocytes assist humoral immunity. At present, great progres and the activity of immunocytes such as peripheral has been made in the research of chemical medicine lood lymphocyte and macrophage are in a low or immunopromoters that play a certain active role highly suppressive state. To effectively solve the pro- in the healthy development of animal husbandry blem, it is necessary to activate the immune system However, these promoters have side or even toxic and increase the transformation rate of the peripheral effects and contain pollution residues. Therefore,re- blood lymphocytes, devouring capability of macro- searchers have been paying more attention to the Received 23 May 2007 Supported by Hebei Science and Technology Office Programme(07220401D) Guangdong Science and Technology Office Programme(2006B203010 1020) SHI Qiumei(1965-), female, Ph. D candidate, professor, engaged in the research of animal infectious disease E-mail: xuebaoenglisheneau. edu.cn C1994-2008ChinaAcademicJOurnalElectronicPublishingHouse.Allrightsreservedhttp:/www.cnki.net
Effects of Immunopotentiator of the Traditional Chinese Medicine on T Lymphocytes in Chicken Blood SHI Qiumei1 , LI Chunling2 , GAO Guisheng1 , SHEN Ping1 , and TANG Shengling1 1 Department of Animal Sciences, Hebei Normal University of Science and Technology, Changli 066600, Hebei, China 2 Veterinary Research Institute, Guangdong Provincial Academy of Agricultural Sciences, Guangzhou 510640, China Abstract：In order to investigate the mechanism of action of immunoenhancer, the effects of the traditional Chinese medicine immunopromoter on the quantity and the transformation rates of T lymphocytes in the chicken blood were determined. Total 120 chickens were randomly assigned into three groups. The 1% and the 0.5% of the Chinese medicine immunopromoter were added to the chicken drinking water, respectively. The quantity of T lymphocytes in each group was measured by a-Naphthyl acetate esterase (ANAE) staining. The results showed that the percentages of T lymphocytes of the treatment groups were significantly higher than that of the control group (P<0.05), and the percentage of the 1% group significantly higher than that of the 0.5% group (P<0.05). In conclusion, the transformation rates of T lymphocytes showed that the Chinese medicine immunopromoter had the significant enhancing effect on the transformation rates of T lymphocytes of the treated chickens. The traditional Chinese medicine immunopromoter had the distinct function to promote the quantity and the transformation rate of T lymphocytes. Key words：traditional Chinese medicine immunopromoter, T lymphocyte, a-Naphthyl acetate esterase staining CLC number: S831; S852.16 Document code: A Article ID: 1006-8104(2008)-02-0026 -04 Introduction There are still outbreaks of infectious diseases in vaccinated chickens and the gradually increasing dosage fails to achieve the ideal therapeutic effects. The reason may be the decreasing immune function of the body due to the long-term mindless use of medicine and unreasonable immune vaccination, especially when the development of lymphocytes and the activity of immunocytes such as peripheral blood lymphocyte and macrophage are in a low or highly suppressive state[1]. To effectively solve the problem, it is necessary to activate the immune system and increase the transformation rate of the peripheral blood lymphocytes, devouring capability of macrophages so that the body can obtain the high-level immune response after vaccination and strengthen the resistance to diseases. The immune function of the animal directly affects its resistance to diseases and the immune effect after vaccination. The level of the immune function of the animal is closely related to the quantity of T and B lymphocytes. It is generally accepted that the function of T lymphocytes can produce lymphokines to increase the immunity and assist humoral immunity. At present, great progress has been made in the research of chemical medicine immunopromoters that play a certain active role in the healthy development of animal husbandry. However, these promoters have side or even toxic effects and contain pollution residues. Therefore, researchers have been paying more attention to the Received 23 May 2007 Supported by Hebei Science and Technology Office Programme (07220401D); Guangdong Science and Technology Office Programme (2006B203010 1020) SHI Qiumei (1965-), female, Ph. D candidate, professor, engaged in the research of animal infectious disease. June 2008 Journal of Northeast Agricultural University Vol. 15 No.2 26-29 E-mail: xuebaoenglish@neau.edu.cn
SHI Qiumei et al. Effects of Immunopotentiator of the Traditional Chinese Medicine on T Lymphocytes in Chicken Blood traditional Chinese medicine immunopromoters2-7. aNaE positive lymphocytes and ANaE negative ones Based on the modern theory of traditional Chinese The anae positive lymphocytes were round with less medicine and herbal medicines and the scientific cytoplasm, and contained a big round green nucleus combination use, we developed the compound called and one or more thick red or brown red granules on Chinese herbal medicine immunopromoter(Astragalus the cytoplasm margin and other parts. The aNAe membranaceus, dangshen, tuckahoe, Rhizome atract- negative lymphocytes were the same as described ylodis macrocephalae and Herba oldenlandiae)and above, but without distinct stained granules. Total 200 its effect on the quantity and the transformation rates lymph of T lymphocytes of chickens by different dosage in calculate the percentage of the ANAe positive lym- drinking water were tested. The aim of this study is to phocytes provide the rationale for the clinical application of the Chinese herbal medicine immunopromoter Transformation rate of the lymphocytes According to the procedures published previously Materials and methods 200 lymphocytes were examined to calculate the trans- formation ra Chinese herbal medicine immunopromoter The transformation rate=Number of transition type The Chinese medicine immunopromoter was com- and blastoma lymphocytes/total number of lympho- posed of Astragalus membranaceus, dangshen, tuck- cytesx100% ahoe, Rhizome atractylodis macrocephalae and Herba oldenlandiae, purchased from Anguo Chinese Herbal Statistic method Medicines Corp, Hebei, China. The quality of all the Analysis of variance and multiple comparison w herbs met the standards of the Pharmacopoeia of Ani- conducted to compare the differences among three mals of the People's Republic of China(2000 Edition). groups The medicines in the recipe were decocted with water to make 1 gmL primary medicine Results and Analysis Animals Quantity of T lymphocytes in blood The chickens were provided by the Animal Farm of The quantity of T lymphocytes in blood is shown Hebei Normal University of Science and Technology, in Table I and Fig. 1. After chickens were fed with and they were randomly assigned into three groups Chinese herbal medicine immunopromoter for 12 days, (n=40 per group) at the age of 12 days. In the drink- the percentage of T lymphocytes of 1% group was ing water for the treatment groups, 1% and 0.5% significantly higher than that of the control(P0.05)or between 0.5% group espectively. Normal drinking water was provided to and control(P>0.05). After chickens were fed with the chickens in the control group Chinese medicine immunopromoter for 24 and 36 significant differe ANAE reaction tage of T lymphocytes among 1% group and control The separation, production, fixing and staining of and 0.5% group, and also between 0.5% group and the lymphocytes were conducted according to the control(P<0.05). After the chickens were fed with procedures published previously. Briefly, after stain- Chinese medicine immunopromoter for 48 days, the e lymphocytes were classified into two types, the percentage of T lymphocytes of 1% group was signi- http://publish.nealeducn C1994-2008ChinaAcademicJOurnalElectronicPublishingHouse.Allrightsreservedhttp:/www.cnki.net
traditional Chinese medicine immunopromoters[2-7]. Based on the modern theory of traditional Chinese medicine and herbal medicines and the scientific combination use, we developed the compound called Chinese herbal medicine immunopromoter (Astragalus membranaceus, dangshen, tuckahoe, Rhizome atractylodis macrocephalae and Herba oldenlandiae) and its effect on the quantity and the transformation rates of T lymphocytes of chickens by different dosage in drinking water were tested. The aim of this study is to provide the rationale for the clinical application of the Chinese herbal medicine immunopromoter. Materials and Methods Chinese herbal medicine immunopromoter The Chinese medicine immunopromoter was composed of Astragalus membranaceus, dangshen, tuckahoe, Rhizome atractylodis macrocephalae and Herba oldenlandiae, purchased from Anguo Chinese Herbal Medicines Corp., Hebei, China. The quality of all the herbs met the standards of the Pharmacopoeia of Animals of the People’s Republic of China (2000 Edition). The medicines in the recipe were decocted with water to make 1 g • mL-1 primary medicine. Animals The chickens were provided by the Animal Farm of Hebei Normal University of Science and Technology, and they were randomly assigned into three groups (n=40 per group) at the age of 12 days. In the drinking water for the treatment groups, 1% and 0.5% concentrations of the Chinese herbal medicine immunopromoter were added to the all-day drinking water, respectively. Normal drinking water was provided to the chickens in the control group. ANAE reaction The separation, production, fixing and staining of the lymphocytes were conducted according to the procedures published previously[8]. Briefly, after staining, the lymphocytes were classified into two types, the ANAE positive lymphocytes and ANAE negative ones. The ANAE positive lymphocytes were round with less cytoplasm, and contained a big round green nucleus and one or more thick red or brown red granules on the cytoplasm margin and other parts. The ANAE negative lymphocytes were the same as described above, but without distinct stained granules. Total 200 lymphocytes were examined under the microscope to calculate the percentage of the ANAE positive lymphocytes. Transformation rate of the lymphocytes According to the procedures published previously[8-9], 200 lymphocytes were examined to calculate the transformation rate using the following formula: The transformation rate=Number of transition type and blastoma lymphocytes/total number of lymphocytes×100%. Statistic method Analysis of variance and multiple comparison was conducted to compare the differences among three groups. Results and Analysis Quantity of T lymphocytes in blood The quantity of T lymphocytes in blood is shown in Table 1 and Fig. 1. After chickens were fed with Chinese herbal medicine immunopromoter for 12 days, the percentage of T lymphocytes of 1% group was significantly higher than that of the control (P0.05) or between 0.5% group and control (P>0.05). After chickens were fed with Chinese medicine immunopromoter for 24 and 36 days, there was a significant difference in the percentage of T lymphocytes among 1% group and control and 0.5% group, and also between 0.5% group and control (P<0.05). After the chickens were fed with Chinese medicine immunopromoter for 48 days, the percentage of T lymphocytes of 1% group was signihttp: //publish.neau.edu.cn SHI Qiumei et al. Effects of Immunopotentiator of the Traditional Chinese Medicine on T Lymphocytes in Chicken Blood ·27·
Journal of Northeast Agricultural University vol.15No.22008 ficantly higher than that of the control (P<0.01)and group was also significantly higher than that of control 0.5% group(P<0.01), and the percentage of 0.5% (P<0.01) Table 1 Effect of the Chinese herbal medicine immunopromoter on the quantity of T lymphocytes in blood (% Days after treatment(d) 0.22+005D 0.35±0.05BD 0.36±0.05BD 0.39±0.05Aa 0.5% 0.23+0.05b 0.23±0.04b 0.28±0.05d Control 0.15±0.0 0.18±0.02 0.19±0.02 0.21±0.09 Different small letters indicate significant difference(P< 0.05), different capital letters indicate extremely significant difference(P<0.01). The same as below cytes is summarized in Table 2 and Fig. 2. The trans- formation rates of T lymphocytes of 0.5% and 1% groups were 83.58% and 88.2 1% respectively, whereas it was 77.90% for control. There were significant differences among the groups(P<0. 05) After chickens were fed with chinese medicine immunopromoter for 24 and 36 days, there was a very Fig 1 ANAE stained T lymphocytes significant difference between control and 1% or 0.5% 1. ANAE stain positive T lymphocyte; 2, ANAE stain negative T group(P<0.01) lymphocytes. The arrows indicate ANAE stained granules After treatment for 30 and 48 days, there was a significant difference between 1% and 0.5% groups Transformation rates of T lymphocytes (P<0.05)and a very significant difference between The result of the transformation rates of T lympho- 0.5% group and control (P<0.01) Table 2 Transformation rates of T lymphocytes(x+s)% after treatments Days after treatment(d) 87.56±0.75A 89.34±1.25Aa 87.50±2.30Aa 88.44±2.40A 0.5% 82.53±2.13B 8400±0.62BbA 84.70±0.74Bb 84.78±1.15B 78.50±0.52D 78.78±0.69D 7831±0.92D 9.25±0.42D Fig. 2 Development of transformation rate of T lymphocytes in different periods 1, Lymphoctoblast; 2. Transitional lymphocyte: 3. Untransitional lymphocyte E-mail: xuebaoenglisheneau. edu.cn C1994-2008ChinaAcademicJournalElectronicPublishingHouse.Allrightsreservedhttp://www.cnki.net
Group Days after treatment (d) 24 36 48 60 1% 87.56±0.75 A 89.34±1.25 Aa 87.50±2.30 Aa 88.44±2.40 A 0.5% 82.53±2.13 B 84.00±0.62 BbA 84.70±0.74 Bb 84.78±1.15 B Control 78.50±0.52 D 78.78±0.69 D 78.31±0.92 D 79.25±0.42 D Transformation rates of T lymphocytes The result of the transformation rates of T lymphocytes is summarized in Table 2 and Fig. 2. The transformation rates of T lymphocytes of 0.5% and 1% groups were 83.58% and 88.21% respectively, whereas it was 77.90% for control. There were significant differences among the groups (P<0.05). After chickens were fed with Chinese medicine immunopromoter for 24 and 36 days, there was a very significant difference between control and 1% or 0.5% group (P<0.01). After treatment for 30 and 48 days, there was a significant difference between 1% and 0.5% groups (P<0.05) and a very significant difference between 0.5% group and control (P<0.01). ficantly higher than that of the control (P<0.01) and 0.5% group (P<0.01), and the percentage of 0.5% group was also significantly higher than that of control (P<0.01). Group Days after treatment (d) 0 12 24 36 48 1% 0.22±0.05 D 0.35±0.05 BD 0.36±0.05 BD 0.39±0.05 Aa 0.5% 0.18±0.04 0.23±0.05 b 0.23±0.04 b 0.28±0.05 d Control 0.15±0.02 0.18±0.02 0.19±0.02 0.21±0.09 Table 1 Effect of the Chinese herbal medicine immunopromoter on the quantity of T lymphocytes in blood (%) l l 2 Fig. 1 ANAE stained T lymphocytes 1, ANAE stain positive T lymphocyte; 2, ANAE stain negative T lymphocytes. The arrows indicate ANAE stained granules. Table 2 Transformation rates of T lymphocytes (x ± s)% after treatments Different small letters indicate significant difference (P< 0.05), different capital letters indicate extremely significant difference (P<0.01). The same as below. l 2 2 3 Fig. 2 Development of transformation rate of T lymphocytes in different periods 1, Lymphoctoblast; 2, Transitional lymphocyte; 3. Untransitional lymphocyte. E-mail: xuebaoenglish@neau.edu.cn ·28· Journal of Northeast Agricultural University Vol. 15 No.2 2008
SHI Qiumei et al. Effects of Immunopotentiator of the Traditional Chinese Medicine on T Lymphocytes in Chicken Blood cells of chickens so as to raise the cell immunity and hu- Discussion moral immune function. If it is applied to the chicken industry, it would play an important role in increasing In the immunocompetent cells of the poultry, the quan- the non-specific immunity and specific immunity of tity of T lymphocytes, secretory lymph and the func- chickens and have a greater practical production value tion of lymphokines directly determine the effect of the cellular immunity of the body. Hence the number References of T lymphocytes is the representative of the degree of I Wang SR, Wang XL, Han WY. Modern animal the immune function. The published data showed that IM. 2nd ed. Changchun: Jilin Science Technology Press, 2001 the polysaccharide can promote thymus development, 523-527 delay thymus atrophy, increase the quantity of T 2 Wang F C, Han Y C, Wang C x, et al. Preliminary report of lymphocytes, and maintain the specific and non research on veterinary Chinese herbal medicine highly-effective specific immune function 2 71, 1o-nll In this study, the immunopromoter [). Shanxi Agricultural Science, 2001, 29: 74-80 quantity of T lymphocytes of the treated chickens in- 3 Ding QL, Cal JQ. Overview of Chinese herbal medicine immune creased continuously between the ages of 24 and 60 research []. Heilongjiang Journal of Animal Husbandry and veteri- days after they were fed with the Chinese herbal me- nary,1998(7):43-44 dicine immunopromoter at the age of 12 days. There 4 ChenS Y, Yu F S. The immunopromoter in the application of anl- were significant differences in the quantity of T lym mal and poultry immunity P]. Fujian Journal of Animal Husbandry phocytes between each group at the ages of 36 and 48 and Veterinary, 1995(3): 14-15 days, and very significant differences between each 5 Shan G Q, Gu xL, Wang W, et al. The effect of Chinese herbal group at the ages of 60 days. This demonstrates that the medicine immunopromoter on immune function of chickens J] use of the Chinese herbal medicine immunopromoter Poultry Husbandry and Diseases Control, 1998(4): 11-12. in the study significantly increases the quantity of T 6 Lu cD, Fang Y H. The effect of compound Fructus Ligustri Lucidi- lymphocytes in the body, and the quantity of T lym us membranaceus preparation on increasing immunity of phocytes is in direct proportion to the concentration of chickens P ]. Chinese Journal of Veterinary Medicine, 2003(1): 30 drinking immunopromoter 7 Zhang Q J, Liu X D. The effect of increasing immunity powder The quantity of T lymphocytes with immunocom- on the immune function and weight gain of chickens [].Chinese petence, secretory lymph and the function of lympho- Joumal of veterinary Medicine, 2002(1 ):35 kines in the immunocompetent cells in the poultry 8 BiKD. Cui s j. research on the effect of Chinese herbal medicine body directly determine the effect of the cell immuni olysaccharide on the transformation rate of lymphocytes and of the body. In this study, the transformation rate percentage of ANAE lymphocytes of chickens P]. Chinese Journal changes of T lymphocytes of the control indicate that of Preventive Veterinary Medicine, 2000, 22: 290-292. the immune function of chicken developes contin- 9 Tang XL, Hu Y L, Song D L, et al. The effect of Astragalus poly- uously with the increase of the age. The transformation saccharide on transformation function of peripheral blood T lympho- rate of 1% concentration group was better than that cytes U ]. Chinese Journal of Veterinary Science, 1998, 18: 269-27 of 0.5% group. The results of the transformation rate 10 Cheng X C, Zhang C J. Research on the effect of Chinese herbal indicate that the Chinese herbal medicine immuno- medicine immunopromoter on immune organ growth and develop- promoter can significantly promote the transformation munocompetent cells of chickens P ]. Chinese Journal rate of T lymphocytes and quicken the development of of Veterinary Medicine, 2002(3): 6-8. immune function of lymphocytes I1 Hu G J, Bai H Q. The regulating effect of Chinese wolfberry on The traditional Chinese medicine imm multiplication of T, B lymphocytes and change of T lymphocytes can promote the multiplication of immunocompetent subgroup ] Chinese Journal of Immunology, 1995(11): 163-166. http://publish.nealeducn C1994-2008ChinaAcademicJOurnalElectronicPublishingHouse.Allrightsreservedhttp:/www.cnki.net
Discussion In the immunocompetent cells of the poultry, the quantity of T lymphocytes, secretory lymph and the function of lymphokines directly determine the effect of the cellular immunity of the body. Hence the number of T lymphocytes is the representative of the degree of the immune function. The published data showed that the polysaccharide can promote thymus development, delay thymus atrophy, increase the quantity of T lymphocytes, and maintain the specific and nonspecific immune function[2, 5-7, 10-11]. In this study, the quantity of T lymphocytes of the treated chickens increased continuously between the ages of 24 and 60 days after they were fed with the Chinese herbal medicine immunopromoter at the age of 12 days. There were significant differences in the quantity of T lymphocytes between each group at the ages of 36 and 48 days, and very significant differences between each group at the ages of 60 days. This demonstrates that the use of the Chinese herbal medicine immunopromoter in the study significantly increases the quantity of T lymphocytes in the body, and the quantity of T lymphocytes is in direct proportion to the concentration of drinking immunopromoter. The quantity of T lymphocytes with immunocompetence, secretory lymph and the function of lymphokines in the immunocompetent cells in the poultry body directly determine the effect of the cell immunity of the body. In this study, the transformation rate changes of T lymphocytes of the control indicate that the immune function of chicken developes continuously with the increase of the age. The transformation rate of 1% concentration group was better than that of 0.5% group. The results of the transformation rate indicate that the Chinese herbal medicine immunopromoter can significantly promote the transformation rate of T lymphocytes and quicken the development of immune function of lymphocytes. The traditional Chinese medicine immunopromoter can promote the multiplication of immunocompetent cells of chickens so as to raise the cell immunity and humoral immune function. If it is applied to the chicken industry, it would play an important role in increasing the non-specific immunity and specific immunity of chickens and have a greater practical production value. References 1 Wang S R, Wang X L, Han W Y. Modern animal immunology [M]. 2nd ed. Changchun: Jilin Science & Technology Press, 2001: 523-527. 2 Wang F C, Han Y C, Wang C X, et al. Preliminary report of research on veterinary Chinese herbal medicine highly-effective immunopromoter [J]. Shanxi Agricultural Science, 2001, 29: 74-80. 3 Ding Q L, Cai J Q. Overview of Chinese herbal medicine immune research [J]. Heilongjiang Journal of Animal Husbandry and Veterinary, 1998(7): 43- 44. 4 Chen S Y, Yu F S. The immunopromoter in the application of animal and poultry immunity [J]. Fujian Journal of Animal Husbandry and Veterinary, 1995(3): 14-15. 5 Shan G Q, Gu X L, Wang W, et al. The effect of Chinese herbal medicine immunopromoter on immune function of chickens [J]. Poultry Husbandry and Diseases Control, 1998(4): 11-12. 6 Lu C D, Fang Y H. The effect of compound Fructus Ligustri LucidiAstragalus membranaceus preparation on increasing immunity of chickens [J]. Chinese Journal of Veterinary Medicine, 2003(1): 30. 7 Zhang Q J, Liu X D. The effect of increasing immunity powder on the immune function and weight gain of chickens [J]. Chinese Journal of Veterinary Medicine, 2002(1): 35. 8 Bi K D, Cui S J. Research on the effect of Chinese herbal medicine polysaccharide on the transformation rate of lymphocytes and percentage of ANAE lymphocytes of chickens [J]. Chinese Journal of Preventive Veterinary Medicine, 2000, 22: 290-292. 9 Tang X L, Hu Y L, Song D L, et al. The effect of Astragalus polysaccharide on transformation function of peripheral blood T lymphocytes [J]. Chinese Journal of Veterinary Science, 1998, 18: 269-271. 10 Cheng X C, Zhang C J. Research on the effect of Chinese herbal medicine immunopromoter on immune organ growth and development and immunocompetent cells of chickens [J]. Chinese Journal of Veterinary Medicine, 2002(3): 6-8. 11 Hu G J, Bai H Q. The regulating effect of Chinese wolfberry on multiplication of T, B lymphocytes and change of T lymphocytes subgroup [J]. Chinese Journal of Immunology, 1995(11): 163-166. http: //publish.neau.edu.cn SHI Qiumei et al. Effects of Immunopotentiator of the Traditional Chinese Medicine on T Lymphocytes in Chicken Blood ·29·