Clearance of Enteric Salmonella enterica Serovar Typhimurium in Chickens Is Independent of B-Cell Function
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感染与免疫杂志 2006年第2期
Divisions of Immunology Microbiology, Institute for Animal Health, Compton, Newbury, Berkshire, United Kingdom
ABSTRACT
Salmonella enterica serovar Typhimurium colonizes the gut of chickens and is cleared from the intestine within about 3 weeks. Infection induces high levels of specific antibody, but B cells do not play an essential role in clearance of primary infection or in the enhanced clearance after secondary challenge.
TEXT
Consumption of contaminated poultry meat and eggs is a major cause of human salmonellosis, with the vast majority of these cases caused by Salmonella enterica serovar Enteritidis or Typhimurium. In chickens these serovars colonize the gastrointestinal tract, with only low-level systemic involvement (4). High levels of Salmonella-specific immunoglobulin M (IgM), IgG, and IgA have been reported that coincide with clearance of salmonellae from the gut lumen (5, 7, 13, 14).
B-cell-deficient chickens have been produced experimentally by surgical removal of the bursa of Fabricius or by chemical (cyclophosphamide) or hormonal (testosterone) treatment (reviewed in reference 22). The use of chemical or hormonal treatment is effective at B-cell removal but also transiently affects other cell types, including the gut epithelial cells, bone marrow cells, T cells, and thrombocytes (11, 16, 18-20). Chemical and/or hormonal treatments decrease the capacity to clear enteric Salmonella infection in the chicken (1, 9, 10), and these results have been interpreted as evidence for the involvement of B cells in immunity. While these treatments are effective in producing B-cell-deficient chickens, our data indicate that there are differential effects on the biology of serovar Typhimurium infection depending on the method employed.
The course of infection was compared in intact, surgically or chemically bursectomized line 61 chickens at 6 weeks old. Surgical bursectomy was achieved according to the method of Glick and Olah (12) by removal of the bursa at 17 days of embryonic development. Chemical ablation of B cells was achieved by daily intramuscular injection of 3 mg cyclophosphamide during the first 4 days posthatch (19). Chickens were reared as described previously (5), and all groups were challenged orally with 2 x 108 CFU of naladixic acid-resistant serovar Typhimurium F98 (24) at 6 weeks of age. Infection was monitored by plating cloacal swabs onto brilliant green agar supplemented with 20 μg/ml naladixic acid and 1 μg/ml novobiocin as described previously (5). Following incubation (24 h, 37°C), plates were scored using a modified version of the system described by Smith and Tucker (24) (Table 1).
The course of infection was identical with intact and surgically bursectomized chickens (Fig. 1). In contrast, chickens rendered B cell deficient by posthatch treatment with cyclophosphamide had significantly greater numbers of salmonellae than both of the other groups between 1 and 41 days postinfection (dpi) (Fig. 1, representative of three separate experiments). Moreover, most intact and surgically bursectomized chickens cleared infection by 20 dpi, whereas the cyclophosphamide-treated chickens continued to excrete serovar Typhimurium until 41 dpi. Neither surgical nor cyclophosphamide treatment affected the numbers of salmonellae detected in the spleen or liver (data not shown).
Since there was a differential effect of surgical bursectomy and cyclophosphamide treatment on the magnitude and course of infection with serovar Typhimurium, it was important to examine the effectiveness of the respective treatments. The status of the B-cell compartment was verified by assessment of circulating anti-Salmonella antibodies in serum (taken at 21 dpi) and by fluorescence-activated cell sorting analysis of splenocytes. Antigen-specific enzyme-linked immunosorbent assay was performed using a soluble Salmonella antigen preparation (STAgP) as described previously (6). Intact chickens responded to infection by production of STAgP-specific serum IgM, IgG, and IgA (as reported previously (5), whereas no antibody could be detected in either surgically bursectomized or cyclophosphamide-treated chickens (Fig. 2A). Phycoerythrin-labeled anti-BU-1 (Cambridge Bioscience, Cambridge, United Kingdom) recognizes chicken B cells (23, 25) and was used for fluorescence-activated cell sorting analysis. As expected, both surgical bursectomy and cylophosphamide treatment effectively removed B cells from the spleen (0.34% and 0.63% Bu1+ cells, respectively, compared with 19.80% with intact animals) (Fig. 2B).
The differential outcome of infection with cyclophosphamide-treated or surgically bursectomized birds may have been due to chemical disturbance of T-cell responses to serovar Typhimurium antigens. This was examined using a standard [3H]thymidine proliferation assay with splenocytes taken at 48 dpi exposed to antigen (STAgP, 8.1 μg/ml), mitogen (phytohemagglutinin, 20 μg/ml), or unsupplemented medium as described elsewhere (5). Splenocytes from intact or B-cell-deficient chickens proliferated in response to STAgP (proliferation was significantly higher than that with medium alone; P < 0.05) (Fig. 2C) or mitogen (data not shown) with no significant differences according to treatment group. Uninfected birds do not respond to STAgP (data not shown) (5). Supplementation of proliferation assay cultures with irradiated splenocytes (to provide B cells for ex vivo antigen presentation) from infected birds did not alter the capacity of splenocytes from B-cell-deficient chickens to respond to STAgP (data not shown). Although cyclophosphamide treatment transiently affects the T-cell response, these are reported to recover completely by 4 weeks posttreatment (11, 19, 20), and our data confirm this within a Salmonella system.
In the mouse, B cells are not required for the clearance of primary infection with serovar Typhimurium but are involved in immunity to secondary infection (21). Although the systemic nature of serovar Typhimurium infection in mice is quite different from the enteric localization in the chicken, it was appropriate to rechallenge the B-cell-deficient chickens. Following clearance of the primary infection (67 dpi), the intact and B-cell-deficient chickens were rechallenged with spectinomycin-resistant serovar Typhimurium F98 (bacteriologically monitored by cloacal swab using brilliant green agar supplemented with 50 μg/ml spectinomycin). Following rechallenge, all groups cleared the infection more rapidly than they had cleared the primary challenge and at a similar rate irrespective of treatment group. The rechallenged chickens also cleared infection more rapidly than parallel primary (PP) infections in age-matched intact or cyclophosphamide-treated chickens (Fig. 3).
Although substantial antibody responses have been consistently reported in chickens infected with serovar Typhimurium (2, 3, 5, 7, 17), the results presented here indicate that antibodies and B cells are not required to clear either a primary or secondary infection. Our data support the findings of previous studies with cyclophosphamide (1, 10), but the lack of effect of embryonic surgical bursectomy indicates that the effect lies within a non-B-cell compartment. Brownwell et al. (8) used surgical bursectomy to assess the role of the bursa in infection with serovar Typhimurium. Unfortunately, the bursectomy was performed at 8 to 9 days posthatch and would not result in complete B-cell deficiency; therefore, the requirement for B cells could not be determined (15). In our studies, surgical removal of the bursa at 17 days of embryonic development resulted in complete ablation of the B-cell compartment. At present, it is not clear which non-B-cell mechanisms are involved in clearance of primary infection. Moreover, the lack of effect of cyclophosphamide treatment at secondary infection suggests that the mechanism of clearance differs between primary and secondary infection. In the chicken, serovar Typhimurium infection is largely restricted to the gut lumen, and identifying the non-B-cell immune mechanisms that mediate bacterial clearance is important for specific anti-Salmonella vaccine development for chickens and may have implications in the broader context of control of enteric bacterial infection.
ACKNOWLEDGMENTS
We thank the staff of the production and experimental units of the IAH and the Biotechnology and Biological Sciences Research Council, United Kingdom, and DEFRA-HEFCI for funding this research (grant no. 8/BFP11365 and VT-0104).
REFERENCES
1. Arnold, J. W., and P. S. Holt. 1995. Response to Salmonella Enteritidis infection by the immunocompromised avian host. Poult. Sci. 74:656-665.
2. Barrow, P. A. 1992. Further observations on the serological response to experimental Salmonella typhimurium in chickens measured by ELISA. Epidemiol. Infect. 108:231-241.
3. Barrow, P. A., N. Bumstead, K. Marston, M. A. Lovell, and P. Wigley. 2004. Faecal shedding and intestinal colonization of Salmonella enterica in in-bred chickens: the effect of host-genetic background. Epidemiol. Infect. 132:117-126.
4. Barrow, P. A., M. B. Huggins, M. A. Lovell, and J. M. Simpson. 1987. Observations on the pathogenesis of experimental Salmonella typhimurium infection in chickens. Res. Vet. Sci. 42:194-199.
5. Beal, R. K., C. Powers, P. Wigley, P. A. Barrow, and A. L. Smith. 2004. Temporal dynamics of the cellular, humoral and cytokine responses in chickens during primary and secondary infection with Salmonella enterica serovar Typhimurium. Avian Pathol. 33:25-33.
6. Beal, R. K., P. Wigley, C. Powers, S. D. Hulme, P. A. Barrow, and A. L. Smith. 2004. Age at primary infection with Salmonella enterica serovar Typhimurium in the chicken influences persistence of infection and subsequent immunity to re-challenge. Vet. Immunol. Immunopathol. 100:151-164.
7. Brito, J. R., M. Hinton, C. R. Stokes, and G. R. Pearson. 1993. The humoral and cell mediated immune response of young chicks to Salmonella typhimurium and S. Kedougou. Br. Vet. J. 149:225-234.
8. Brownwell, J. R., W. W. Sadler, and M. J. Fanelli. 1970. Role of Bursa of Fabricius in chicken resistance to Salmonella Typhimurium. Avian Dis. 14:142-152.
9. Corrier, D. E., M. H. Elissalde, R. L. Ziprin, and J. R. DeLoach. 1991. Effect of immunosuppression with cyclophosphamide, cyclosporin, or dexamethasone on Salmonella colonization of broiler chicks. Avian Dis. 35:40-45.
10. Desmidt, M., R. Ducatelle, J. Mast, B. M. Goddeeris, B. Kaspers, and F. Haesebrouck. 1998. Role of the humoral immune system in Salmonella enteritidis phage type four infection in chickens. Vet. Immunol. Immunopathol. 63:355-367.
11. Glick, B. 1971. Morphological changes and humoral immunity in cyclophosphamide-treated chicks. Transplantation 11:433-439.
12. Glick, B., and I. Olah. 1984. Methods of bursectomy. Methods Enzymol. 108:3-10.
13. Hassan, J. O., P. A. Barrow, A. P. Mockett, and S. McLeod. 1990. Antibody response to experimental Salmonella typhimurium infection in chickens measured by ELISA. Vet. Rec. 126:519-522.
14. Hassan, J. O., A. P. Mockett, D. Catty, and P. A. Barrow. 1991. Infection and reinfection of chickens with Salmonella typhimurium: bacteriology and immune responses. Avian Dis. 35:809-819.
15. Hemmingsson, E. J., and T. J. Linna. 1972. Ontogenetic studies on lymphoid cell traffic in the chicken. I. Cell migration from the bursa of Fabricius. Int. Arch. Allergy Appl. Immunol. 42:693-710.
16. Kim, Y., T. P. Brown, and M. J. Pantin-Jackwood. 2003. Lesions induced in broiler chickens by cyclophosphamide treatment. Vet. Hum. Toxicol. 45:121-123.
17. Lee, G. M., G. D. Jackson, and G. N. Cooper. 1983. Infection and immune responses in chickens exposed to Salmonella typhimurium. Avian Dis. 27:577-583.
18. Lerman, S. P., and W. P. Weidanz. 1970. The effect of cyclophosphamide on the ontogeny of the humoral immune response in chickens. J. Immunol. 105:614-619.
19. Linna, T. J., D. Frommel, and R. A. Good. 1972. Effects of early cyclophosphamide treatment on the development of lymphoid organs and immunological functions in the chickens. Int. Arch. Allergy Appl. Immunol. 42:20-39.
20. Mast, J., M. Desmidt, G. Room, C. Martin, R. Ducatelle, S. Haesebrouck, T. F. Davison, B. Kaspers, and B. M. Goddeeris. 1997. Different methods of bursectomy induce different effects on leukocyte distribution and reactivity. Arch. Gefluegelk. 61:238-246.
21. Mastroeni, P., C. Simmons, R. Fowler, C. E. Hormaeche, and G. Dougan. 2000. Igh-6–/– (B-cell-deficient) mice fail to mount solid acquired resistance to oral challenge with virulent Salmonella enterica serovar Typhimurium and show impaired Th1 T-cell responses to Salmonella antigens. Infect. Immun. 68:46-53.
22. Ratcliffe, J. H. 1997. The study of B cell differentiation, p. 2199-2212. In I. Lefkowits (ed.), Immunology methods manual, vol. 4. Academic Press Ltd., London, United Kingdom.
23. Rothwell, C. J., L. Vervelde, and T. F. Davison. 1996. Identification of chicken Bu-1 alloantigens using the monoclonal antibody AV20. Vet. Immunol. Immunopathol. 55:225-234.
24. Smith, H. W., and J. F. Tucker. 1975. The effect of antibiotic therapy on the faecal excretion of Salmonella Typhimurium by experimentally infected chickens. J. Hyg. (London) 75:275-292.
25. Tregaskes, C. A., N. Bumstead, T. F. Davison, and J. R. Young. 1996. Chicken B-cell marker chB6 (Bu-1) is a highly glycosylated protein of unique structure. Immunogenetics 44:212-217.(Richard K. Beal, Claire P)
ABSTRACT
Salmonella enterica serovar Typhimurium colonizes the gut of chickens and is cleared from the intestine within about 3 weeks. Infection induces high levels of specific antibody, but B cells do not play an essential role in clearance of primary infection or in the enhanced clearance after secondary challenge.
TEXT
Consumption of contaminated poultry meat and eggs is a major cause of human salmonellosis, with the vast majority of these cases caused by Salmonella enterica serovar Enteritidis or Typhimurium. In chickens these serovars colonize the gastrointestinal tract, with only low-level systemic involvement (4). High levels of Salmonella-specific immunoglobulin M (IgM), IgG, and IgA have been reported that coincide with clearance of salmonellae from the gut lumen (5, 7, 13, 14).
B-cell-deficient chickens have been produced experimentally by surgical removal of the bursa of Fabricius or by chemical (cyclophosphamide) or hormonal (testosterone) treatment (reviewed in reference 22). The use of chemical or hormonal treatment is effective at B-cell removal but also transiently affects other cell types, including the gut epithelial cells, bone marrow cells, T cells, and thrombocytes (11, 16, 18-20). Chemical and/or hormonal treatments decrease the capacity to clear enteric Salmonella infection in the chicken (1, 9, 10), and these results have been interpreted as evidence for the involvement of B cells in immunity. While these treatments are effective in producing B-cell-deficient chickens, our data indicate that there are differential effects on the biology of serovar Typhimurium infection depending on the method employed.
The course of infection was compared in intact, surgically or chemically bursectomized line 61 chickens at 6 weeks old. Surgical bursectomy was achieved according to the method of Glick and Olah (12) by removal of the bursa at 17 days of embryonic development. Chemical ablation of B cells was achieved by daily intramuscular injection of 3 mg cyclophosphamide during the first 4 days posthatch (19). Chickens were reared as described previously (5), and all groups were challenged orally with 2 x 108 CFU of naladixic acid-resistant serovar Typhimurium F98 (24) at 6 weeks of age. Infection was monitored by plating cloacal swabs onto brilliant green agar supplemented with 20 μg/ml naladixic acid and 1 μg/ml novobiocin as described previously (5). Following incubation (24 h, 37°C), plates were scored using a modified version of the system described by Smith and Tucker (24) (Table 1).
The course of infection was identical with intact and surgically bursectomized chickens (Fig. 1). In contrast, chickens rendered B cell deficient by posthatch treatment with cyclophosphamide had significantly greater numbers of salmonellae than both of the other groups between 1 and 41 days postinfection (dpi) (Fig. 1, representative of three separate experiments). Moreover, most intact and surgically bursectomized chickens cleared infection by 20 dpi, whereas the cyclophosphamide-treated chickens continued to excrete serovar Typhimurium until 41 dpi. Neither surgical nor cyclophosphamide treatment affected the numbers of salmonellae detected in the spleen or liver (data not shown).
Since there was a differential effect of surgical bursectomy and cyclophosphamide treatment on the magnitude and course of infection with serovar Typhimurium, it was important to examine the effectiveness of the respective treatments. The status of the B-cell compartment was verified by assessment of circulating anti-Salmonella antibodies in serum (taken at 21 dpi) and by fluorescence-activated cell sorting analysis of splenocytes. Antigen-specific enzyme-linked immunosorbent assay was performed using a soluble Salmonella antigen preparation (STAgP) as described previously (6). Intact chickens responded to infection by production of STAgP-specific serum IgM, IgG, and IgA (as reported previously (5), whereas no antibody could be detected in either surgically bursectomized or cyclophosphamide-treated chickens (Fig. 2A). Phycoerythrin-labeled anti-BU-1 (Cambridge Bioscience, Cambridge, United Kingdom) recognizes chicken B cells (23, 25) and was used for fluorescence-activated cell sorting analysis. As expected, both surgical bursectomy and cylophosphamide treatment effectively removed B cells from the spleen (0.34% and 0.63% Bu1+ cells, respectively, compared with 19.80% with intact animals) (Fig. 2B).
The differential outcome of infection with cyclophosphamide-treated or surgically bursectomized birds may have been due to chemical disturbance of T-cell responses to serovar Typhimurium antigens. This was examined using a standard [3H]thymidine proliferation assay with splenocytes taken at 48 dpi exposed to antigen (STAgP, 8.1 μg/ml), mitogen (phytohemagglutinin, 20 μg/ml), or unsupplemented medium as described elsewhere (5). Splenocytes from intact or B-cell-deficient chickens proliferated in response to STAgP (proliferation was significantly higher than that with medium alone; P < 0.05) (Fig. 2C) or mitogen (data not shown) with no significant differences according to treatment group. Uninfected birds do not respond to STAgP (data not shown) (5). Supplementation of proliferation assay cultures with irradiated splenocytes (to provide B cells for ex vivo antigen presentation) from infected birds did not alter the capacity of splenocytes from B-cell-deficient chickens to respond to STAgP (data not shown). Although cyclophosphamide treatment transiently affects the T-cell response, these are reported to recover completely by 4 weeks posttreatment (11, 19, 20), and our data confirm this within a Salmonella system.
In the mouse, B cells are not required for the clearance of primary infection with serovar Typhimurium but are involved in immunity to secondary infection (21). Although the systemic nature of serovar Typhimurium infection in mice is quite different from the enteric localization in the chicken, it was appropriate to rechallenge the B-cell-deficient chickens. Following clearance of the primary infection (67 dpi), the intact and B-cell-deficient chickens were rechallenged with spectinomycin-resistant serovar Typhimurium F98 (bacteriologically monitored by cloacal swab using brilliant green agar supplemented with 50 μg/ml spectinomycin). Following rechallenge, all groups cleared the infection more rapidly than they had cleared the primary challenge and at a similar rate irrespective of treatment group. The rechallenged chickens also cleared infection more rapidly than parallel primary (PP) infections in age-matched intact or cyclophosphamide-treated chickens (Fig. 3).
Although substantial antibody responses have been consistently reported in chickens infected with serovar Typhimurium (2, 3, 5, 7, 17), the results presented here indicate that antibodies and B cells are not required to clear either a primary or secondary infection. Our data support the findings of previous studies with cyclophosphamide (1, 10), but the lack of effect of embryonic surgical bursectomy indicates that the effect lies within a non-B-cell compartment. Brownwell et al. (8) used surgical bursectomy to assess the role of the bursa in infection with serovar Typhimurium. Unfortunately, the bursectomy was performed at 8 to 9 days posthatch and would not result in complete B-cell deficiency; therefore, the requirement for B cells could not be determined (15). In our studies, surgical removal of the bursa at 17 days of embryonic development resulted in complete ablation of the B-cell compartment. At present, it is not clear which non-B-cell mechanisms are involved in clearance of primary infection. Moreover, the lack of effect of cyclophosphamide treatment at secondary infection suggests that the mechanism of clearance differs between primary and secondary infection. In the chicken, serovar Typhimurium infection is largely restricted to the gut lumen, and identifying the non-B-cell immune mechanisms that mediate bacterial clearance is important for specific anti-Salmonella vaccine development for chickens and may have implications in the broader context of control of enteric bacterial infection.
ACKNOWLEDGMENTS
We thank the staff of the production and experimental units of the IAH and the Biotechnology and Biological Sciences Research Council, United Kingdom, and DEFRA-HEFCI for funding this research (grant no. 8/BFP11365 and VT-0104).
REFERENCES
1. Arnold, J. W., and P. S. Holt. 1995. Response to Salmonella Enteritidis infection by the immunocompromised avian host. Poult. Sci. 74:656-665.
2. Barrow, P. A. 1992. Further observations on the serological response to experimental Salmonella typhimurium in chickens measured by ELISA. Epidemiol. Infect. 108:231-241.
3. Barrow, P. A., N. Bumstead, K. Marston, M. A. Lovell, and P. Wigley. 2004. Faecal shedding and intestinal colonization of Salmonella enterica in in-bred chickens: the effect of host-genetic background. Epidemiol. Infect. 132:117-126.
4. Barrow, P. A., M. B. Huggins, M. A. Lovell, and J. M. Simpson. 1987. Observations on the pathogenesis of experimental Salmonella typhimurium infection in chickens. Res. Vet. Sci. 42:194-199.
5. Beal, R. K., C. Powers, P. Wigley, P. A. Barrow, and A. L. Smith. 2004. Temporal dynamics of the cellular, humoral and cytokine responses in chickens during primary and secondary infection with Salmonella enterica serovar Typhimurium. Avian Pathol. 33:25-33.
6. Beal, R. K., P. Wigley, C. Powers, S. D. Hulme, P. A. Barrow, and A. L. Smith. 2004. Age at primary infection with Salmonella enterica serovar Typhimurium in the chicken influences persistence of infection and subsequent immunity to re-challenge. Vet. Immunol. Immunopathol. 100:151-164.
7. Brito, J. R., M. Hinton, C. R. Stokes, and G. R. Pearson. 1993. The humoral and cell mediated immune response of young chicks to Salmonella typhimurium and S. Kedougou. Br. Vet. J. 149:225-234.
8. Brownwell, J. R., W. W. Sadler, and M. J. Fanelli. 1970. Role of Bursa of Fabricius in chicken resistance to Salmonella Typhimurium. Avian Dis. 14:142-152.
9. Corrier, D. E., M. H. Elissalde, R. L. Ziprin, and J. R. DeLoach. 1991. Effect of immunosuppression with cyclophosphamide, cyclosporin, or dexamethasone on Salmonella colonization of broiler chicks. Avian Dis. 35:40-45.
10. Desmidt, M., R. Ducatelle, J. Mast, B. M. Goddeeris, B. Kaspers, and F. Haesebrouck. 1998. Role of the humoral immune system in Salmonella enteritidis phage type four infection in chickens. Vet. Immunol. Immunopathol. 63:355-367.
11. Glick, B. 1971. Morphological changes and humoral immunity in cyclophosphamide-treated chicks. Transplantation 11:433-439.
12. Glick, B., and I. Olah. 1984. Methods of bursectomy. Methods Enzymol. 108:3-10.
13. Hassan, J. O., P. A. Barrow, A. P. Mockett, and S. McLeod. 1990. Antibody response to experimental Salmonella typhimurium infection in chickens measured by ELISA. Vet. Rec. 126:519-522.
14. Hassan, J. O., A. P. Mockett, D. Catty, and P. A. Barrow. 1991. Infection and reinfection of chickens with Salmonella typhimurium: bacteriology and immune responses. Avian Dis. 35:809-819.
15. Hemmingsson, E. J., and T. J. Linna. 1972. Ontogenetic studies on lymphoid cell traffic in the chicken. I. Cell migration from the bursa of Fabricius. Int. Arch. Allergy Appl. Immunol. 42:693-710.
16. Kim, Y., T. P. Brown, and M. J. Pantin-Jackwood. 2003. Lesions induced in broiler chickens by cyclophosphamide treatment. Vet. Hum. Toxicol. 45:121-123.
17. Lee, G. M., G. D. Jackson, and G. N. Cooper. 1983. Infection and immune responses in chickens exposed to Salmonella typhimurium. Avian Dis. 27:577-583.
18. Lerman, S. P., and W. P. Weidanz. 1970. The effect of cyclophosphamide on the ontogeny of the humoral immune response in chickens. J. Immunol. 105:614-619.
19. Linna, T. J., D. Frommel, and R. A. Good. 1972. Effects of early cyclophosphamide treatment on the development of lymphoid organs and immunological functions in the chickens. Int. Arch. Allergy Appl. Immunol. 42:20-39.
20. Mast, J., M. Desmidt, G. Room, C. Martin, R. Ducatelle, S. Haesebrouck, T. F. Davison, B. Kaspers, and B. M. Goddeeris. 1997. Different methods of bursectomy induce different effects on leukocyte distribution and reactivity. Arch. Gefluegelk. 61:238-246.
21. Mastroeni, P., C. Simmons, R. Fowler, C. E. Hormaeche, and G. Dougan. 2000. Igh-6–/– (B-cell-deficient) mice fail to mount solid acquired resistance to oral challenge with virulent Salmonella enterica serovar Typhimurium and show impaired Th1 T-cell responses to Salmonella antigens. Infect. Immun. 68:46-53.
22. Ratcliffe, J. H. 1997. The study of B cell differentiation, p. 2199-2212. In I. Lefkowits (ed.), Immunology methods manual, vol. 4. Academic Press Ltd., London, United Kingdom.
23. Rothwell, C. J., L. Vervelde, and T. F. Davison. 1996. Identification of chicken Bu-1 alloantigens using the monoclonal antibody AV20. Vet. Immunol. Immunopathol. 55:225-234.
24. Smith, H. W., and J. F. Tucker. 1975. The effect of antibiotic therapy on the faecal excretion of Salmonella Typhimurium by experimentally infected chickens. J. Hyg. (London) 75:275-292.
25. Tregaskes, C. A., N. Bumstead, T. F. Davison, and J. R. Young. 1996. Chicken B-cell marker chB6 (Bu-1) is a highly glycosylated protein of unique structure. Immunogenetics 44:212-217.(Richard K. Beal, Claire P)