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Salmonella enteritidis clearance and immune responses in chickens following Salmonella vaccination and challenge
Veterinary Immunology and Immunopathology 101 (2004) 251–257
Salmonella enteritidis clearance and immune responses in chickens following Salmonella vaccination and challenge U. Babua,*, R.A. Dalloulb,c, M. Okamurab, H.S. Lillehojb, H. Xiec, R.B. Raybournea, D. Gainesa, R.A. Heckertc a
Center for Food Safety and Applied Nutrition, US Food and Drug Administration, HFS-326, 8301 Muirkirk Road, Laurel, MD 20708, USA b Animal Parasitic Diseases Laboratory, Animal and Natural Resources Institute, ARS, USDA, Building 1040 BARC-East, Beltsville, MD 20705, USA c Virginia–Maryland Regional College of Veterinary Medicine, University of Maryland, 8075 Greenmead Drive, College Park, MD 20742, USA Received 29 October 2003; received in revised form 6 May 2004; accepted 20 May 2004
Abstract Our previous work showed that the cell-mediated immunity (CMI) was enhanced by live Salmonella vaccine (LV). The objective of this study was to evaluate the impact of live and killed Salmonella vaccines on Salmonella enteritidis (SE) clearance and to determine if the clearance was mediated by cell-mediated and/or humoral immunity. Chickens were first immunized at 2 weeks of age followed by a booster dose at 4 weeks, challenged with live SE 2 weeks later (6-week-old) and tested for CMI, antibody response and SE clearance 1-week post SE-challenge (7-week-old). Spleen cell proliferation induced by SE-flagella and Concanavalin A (Con A) were significantly higher and SE shedding was significantly lower in the LV group. The splenic CD3 population was significantly lower and B cells were higher in the control group compared to all the SE-challenged groups (with and without vaccination). Serum antibody to SE-flagella and envelope were significantly higher in the KV group compared to all the other groups. These results suggest that LV protects against SE infection, probably by enhancing the CMI. # 2004 Elsevier B.V. All rights reserved. Keywords: Salmonella enteritidis; Vaccine; Chicken; Flagella; Lymphocyte proliferation
1. Introduction A significant proportion of human salmonellosis is known to be caused by consumption of raw or partially Abbreviations: CMI, cell-mediated immunity; DPI, days post immunization; DTH, delayed type hypersensitivity; HBSS, Hank’s balanced salt solution; h, hour(s); KV, killed vaccine; LV, live vaccine; min, minute; MNC, mononuclear cells; RT, room temperature; SE, Salmonella enteritidis; s, seconds * Corresponding author. Tel.: þ1 301 827 8515; fax: þ1 301 594 0517. E-mail address: [email protected] (U. Babu)
cooked eggs (Hope et al., 2002). On average, Americans consume 234 eggs/person/year. Salmonellaassociated food poisoning can result in significant financial burden on the health care system (Hasenson et al., 1992; Roberts and Sockett, 1994). Therefore, it is important to control egg contamination to reduce the worldwide salmonellosis problem. Besides good hygiene and animal husbandry practices, several methods have been currently employed to reduce Salmonella infection in poultry farms, such as the use of antibiotics, competitive exclusion by nonpathogenic bacteria, genetic selection of chicken strains for
0165-2427/$ – see front matter # 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2004.05.002
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U. Babu et al. / Veterinary Immunology and Immunopathology 101 (2004) 251–257
improved immune response, and development of Salmonella vaccines (Lillehoj et al., 2000). The efficacy of killed and live attenuated vaccine, and crude Salmonella immune lymphokines has been tested using chickens of different ages, various doses and routes of inoculation (Cooper et al., 1994; Hassan and Curtiss, 1990, 1994a, 1996). Generally, the immunopotentiating role of vaccines has been tested by their ability to stimulate antibody responses (Hassan and Curtiss, 1990, 1994a, 1996). Measurement of cell-mediated immunity (CMI) in vaccinated birds has been limited to the delayed type hypersensitivity (DTH) response to Salmonella typhimurium outer membrane proteins (Hassan and Curtiss, 1994b). Moreover, extensive mouse studies have shown the role for T cells, natural killer (NK) cells, macrophages, neutrophils and numerous cytokines in Salmonella resistance (as reviewed by Lillehoj and Okamura, 2003; Mastroeni et al., 2001; Mittrucker and Kaufmann, 2000). Our recently published study showed that a live vaccine (LV) was effective in enhancing the CMI of 18- and 32-week-old chickens as seen by the increased splenic lymphocyte proliferation to Salmonella enteritidis (SE) antigens and Con A (Babu et al., 2003). However, in that study we did not test if the increased CMI would influence SE clearance. Therefore, this study was conducted to evaluate the impact of live and killed vaccines on the ability of chickens to clear SE infection and to elucidate if the bacterial clearance was mediated by an enhanced immune response. Results from these studies showed that the live vaccine was more effective than the killed vaccine in clearing SE, which was accompanied by increased CMI. This may offer useful information for managing SE infection in chickens and shell egg contamination with SE.
2. Materials and methods 2.1. Birds, bacteria and vaccines Specific pathogen free White Leghorn chickens from Hyline International (Dallas Center, IA) were obtained as fertile eggs, hatched at the Animal and Natural Resources Institute facilities, Agricultural Research Service, US Department of Agriculture (Beltsville, MD). Chickens were kept in wire cages
and provided feed and water ad libitum with constant light source until use. One week before use, the birds were moved and housed in individual isolation units in a biosafety level 2 research facility at the College of Veterinary Medicine, University of Maryland (College Park, MD). Birds were provided with water and commercial antibiotic-free feed ad libitum. The experimental procedures and protocols were undertaken in accordance with the standards relating to the animal care and management guidelines of the University of Maryland. Salmonella enterica serovar Enteritidis phage type 4 strain 338 (Sheela et al., 2003) was diluted to 1 1010 CFU/ml in phosphate buffered saline (PBS) before inoculation. The live vaccine (Megan1Vac 1, Megan Health Inc., St. Louis, MO) was a Dcya Dcrp gene modified derivative of Salmonella typhimurium belonging to serogroup B (1, 4, 5, 12:i:1, 2). The killed vaccine (Poulvac1 SE, Ft. Dodge Animal Health Inc., Overland Park, KS) was an inactivated oil emulsion bacterin containing Salmonella enteritidis phage types 4, 8, and 13a belonging to the serogroup D1 (1, 9, 12:g, m:[1,7]). 2.2. Experimental design The objective of this study was to evaluate the impact of live and killed vaccines on SE clearance in young chickens. Twenty-four birds were assigned to four groups, namely the control (no vaccine, no challenge), challenge without immunization, the killed and the live vaccine groups with the SE-challenge. Birds in the vaccine groups were first immunized at 2 weeks of age and given a booster dose at 4 weeks of age. Two weeks later, these birds and those in the unvaccinated challenge group (6-week-old) were challenged with an oral infection of S. enteritidis (oral gavage, 1010 SE 338/bird). These birds were euthanized a week later (7-week-old) for immunological analyses and SE clearance. In this study, unvaccinated challenged birds served as control for the vaccinated and challenged birds. This offered us a way to determine the effect of infection alone or infection and immunization together on the immune response of young chickens. Furthermore, we could assess the efficacy of live and killed vaccine on SE clearance when compared to the unvaccinated challenge group.
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2.3. Flagella preparation from SE Flagella were prepared from Salmonella enteritidis strain 338 (SE 338) as described previously (Babu et al., 2003). Flagella antigen preparation primarily consisted of a single protein (approximately 54,000 Da) as confirmed by Coomassie Blue staining. 2.4. Isolation of splenic mononuclear cells Splenic mononuclear cells were isolated from fresh spleens as described previously (Babu et al., 2003). Cell concentration was adjusted to 8 106 cells/ml and viability was assessed by Trypan blue exclusion. 2.5. Mitogenic reactivity in vitro Splenic lymphocyte response to Con A- and SEflagella was assessed as described by Babu et al. (2003). Data are presented as counts per minute (cpm) from stimulated cultures after subtracting the cpm from non-stimulated cultures. 2.6. Antibodies and flow cytometric analysis Flow cytometric analysis of MNC was carried out as previously described (Sheela et al., 2003). MNC were incubated with R-phycoerythrin-conjugated anti-chicken CD3, and FITC-conjugated anti-chicken IgG (Southern Biotech, Birmingham, AL). Duplicate samples of MNC were stained with R-PE-anti-chicken CD4, and FITC-anti-chicken CD8 (Southern Biotech). R-PE- and FITC-conjugated isotype control samples were also included. Five thousand cells were analyzed using an EPICS Elite flow cytometer (Beckman/Coulter, Hialeah, FL). For gating purposes, the viable MNC population was determined by forward light scatter versus side light scatter analysis. 2.7. ELISA for serum antibodies Sera were tested for antibodies to two different Salmonella antigens, flagella and envelope. Flagella were prepared as described in the previous section. SE envelope was prepared by growing SE 338 in TSBYE broth overnight at 37 8C, the bacteria were washed twice with PBS and resuspended in 1 ml PBS for every 50 ml of original broth used. Cells were sonicated
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three times on ice for 1 min with 30 s of resting period after each sonication. Sonicated samples were centrifuged at 2700 rpm then the supernatant centrifuged at 15,000 rpm, each for 30 min at 4 8C. Flat bottom microtiter plates (Dynex Technologies, Chantilly, VA) were coated with 5.0 mg/well flagella Ag or 2.5 mg/well envelope Ag in 100 ml of 0.1 M carbonate buffer for 18 h at 4 8C. The plates were washed twice with PBS-T and blocking followed using 1% BSA– PBS for 30 min at RT. Plates were then flicked well and 100 ml of 1:100 diluted serum samples added to each well and allowed to incubate for 2 h at RT with continuous gentle shaking. The plates were washed 5 with PBS-T, 100 ml peroxidase-conjugated rabbit anti-chicken IgG mAb (dilution 1:2000; Sigma) were added to each well and allowed to incubate for 40 min at RT with shaking. The plates were washed again, 100 ml of TMB added as above, and the OD read at 450 nm using a plate reader. 2.8. Fecal shedding of SE This was carried out by the most probable number (MPN) estimation. Ceca from the six birds killed in each group were pooled and macerated. Isolation and enumeration of viable counts of SE from pools were made by direct plating of 10-fold dilutions of cecal contents on XLT Salmonella selective agar medium (Difco, Baltimore, MD) employing standard procedures (FDA Bacteriological Analytical Manual, 7th ed.). 2.9. Statistical analyses Data pertaining to proliferation and cell populations were compared between the control and the vaccine groups and among the vaccine groups by conducting one-way analysis of variance (ANOVA) followed by Student–Newman–Keuls test. These statistical analyses were done using Sigma Stat v2.01. Differences between the treatment groups were considered statistically significant at P < 0.05.
3. Results and discussion Chickens treated with LV prior to SE-challenge had reduced SE shedding (most probable number ¼ 20þ/g)
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Fig. 1. ConA (A) and SE-flagella (B) mediated spleen cell proliferation in chickens immunized with killed and live SE vaccines followed by live SE-challenge. Two-week-old chickens were immunized with killed and live SE vaccines (primary immunization), and were re-immunized (secondary immunization) 2 weeks later (age 4 weeks) and challenged 2 weeks later (age 6 weeks). SE infection was carried out with 1010 SE organisms. Birds were euthanized 1 week later (age 7 weeks) to assess splenic cell proliferation. Each bar represents mean S.E.M. (n ¼ 8). Bars without common letter differed (P < 0.05). SE: Salmonella enteritidis; KV: killed vaccine; LV: live vaccine.
compared to the unvaccinated birds or those that received the KV prior to challenge (most probable number ¼ 210þ/g for control and >1100þ/g for KV groups). These results are in agreement with previous reports of greater efficacy of live than the killed Salmonella vaccine in eliminating SE from chickens (Hassan and Curtiss, 1990; Mastroeni et al., 2001; Gast et al., 1993; Harrison et al., 1997). In addition to the increased SE clearance in the LV treated birds, spleen cells from these chickens responded significantly better to Con A and SE-flagella than those from the control and the KV groups Fig. 1A and B). These results are similar to our previous observations in older chickens treated with the same LV (Babu et al., 2003) and those from mouse studies in which attenuated SE and ST resulted in increased DTH and in vitro antigen-specific proliferation of spleen
cells, respectively (George et al., 1987; Villarreal et al., 1992). In contrast to the immunopotentiating effect of the live vaccine, we observed a strong negative effect of killed vaccine on the spleen cell proliferation to Con A. Other researchers have shown lower efficacy or immunosuppression by killed vaccine, compared to a more complete immune response elicited by live vaccine in chickens, mice and healthy human volunteers (George et al., 1987, 1989; Curtiss et al., 1993; Cooper, 1994; Harrison et al., 1997). Our results reestablished the importance of CMI in Salmonella clearance which was demonstrated previously in a mouse model (George et al., 1987; Mittrucker and Kaufmann, 2000; Mastroeni et al., 2001; Lalmanach and Lantier, 1999; Naiki et al., 1999). Infection of 6-week-old chickens with live virulent SE resulted in increased splenic CD3 and reduced B
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Table 1 Impact of live and killed Salmonella vaccine and SE-challenge on spleen T cell subsets and B cells of chickens Group
CD8
Control Control-challenge KV-challenge LV-challenge
36.6 43.2 38.9 40.3
CD4
5.5 3.5 6.1 0.52
15.6 16.5 21 18
CD3
3.7 2.5 5.8 4.7
49.2 56.3 58.2 56.2
B cells
6.1 b 1.1 a 5a 3.2 a
42.9 35.5 32.6 36.3
5.1 1.3 5.1 2.9
a b b b
Data are presented as mean S.D. (n ¼ 6 chickens/group). Two-week-old chickens were immunized with killed and live SE vaccines (primary immunization), and were re-immunized 2 weeks later (4-week-old secondary immunization) and challenged 2 weeks later (6-weekold). Challenge infection was carried out with 1010 SE organisms. Chickens were killed a week after challenge (7 weeks of age) and spleen cells were stained for CD4, CD8, CD3, or s-Igþ B cells. Cell subsets were determined by flow cytometry as described in Section 2. Means with different letters within the same column were different at P ¼ 0.05.
cell populations, with the highest CD3/B ratio in the KV group (Table 1). Although we observed increased CD3 cells in the infected groups, it was difficult to associate this increase with SE clearance due to lack of any significant changes in CD4þ or CD8þ cells (Mittrucker et al., 2002). Reduced B cells in Salmonella infected experimental calves and infants with salmonellosis has been reported previously (Liebler et al., 1994; Stoicheva et al., 1997). Importance of B cells in Salmonella infection has been established by reduced LD50 and impaired resistance to Salmonella typhimuriun in B cell deficient mice and by decreased SE clearance in young chickens treated with testosterone propionate which selectively depletes B cells (Mastroeni et al., 2000; Arnold and Holt, 1995). In our study, although B cell depletion was similar in all the challenged birds, SE clearance was significantly higher in the LV group. This suggests that LV produces factors that can possibly overcome the B cell deficit and clear the SE infection in chickens. This speculation is based on live Salmonella typhimurium induced increase in IFN-g, IL-12, and IL-2 (John et al., 2002; Harrison et al., 1997), which are known to play a role in Salmonella clearance (Dybing et al., 1999; van Deventer, 2000). In contrast to its inhibitory effect on CMI and SE clearance, KV induced highest SE-specific antibody (Fig. 2). It is not clear if this increased antibody response is associated with an increased T to B cell ratio in the KV group (Brieva et al., 1983). Our results suggest that the antibody against SE does not protect the chickens against the SE infection. There is some controversy about the involvement of humoral immune response in protection against Salmonella infection, with some studies showing a positive cor-
relation between antibody response and Salmonella clearance and others showing no effect (Hassan and Curtiss, 1990, 1994b; Begg et al., 1990; Sigwart et al., 1989). Overall, this is one of the few studies showing that oral vaccination with LV enhanced CMI with increased SE clearance in chickens. In contrast to the effects of LV, KV enhanced the humoral immune response, significantly inhibited the CMI and increased the bacterial load in young chickens, thus indicating the importance of CMI in SE clearance. Our results suggest that vaccination with attenuated live Salmonella could be an effective method for
Fig. 2. Antibody response in challenged birds following secondary Salmonella immunization. Sera were tested for Abs against flagella and envelope antigens. Each bar represents the mean of three to six birds. Two-week-old chickens were immunized with killed and live SE vaccines (primary immunization), and were re-immunized (secondary immunization) 2 weeks later (age 4 weeks) and challenged 2 weeks later (age 6 weeks). SE infection was carried out with 1010 SE organisms. Each bar represents mean S.E.M. (n ¼ 6). Bars without common letter differed (P < 0.05). SE: Salmonella enteritidis; KV: killed vaccine; LV: live vaccine.
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controlling Salmonella enteritidis infection in young chickens.
Acknowledgements This work was supported by the grant from USDA National Food Safety Initiative Grant 00-51110-9739. We are grateful to S. Elankumaran, R. Sheela, Elmer Bigley and Terry Gaither for their excellent and valuable support in completion of this project. We would like to thank Sandra Kelly-Aehle from Megan Health Inc., for providing the live attenuated vaccine and Jerry D. Maiers, Senior Poultry Technical Services Veterinarian, Fort Dodge Animal Health, for providing the killed vaccine.
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Sheela, R.R., Babu, U., Mu, J., Elankumaran, S., Bautista, D.A., Raybourne, R.B., Heckert, R.A., Song, W., 2003. Immune responses against Salmonella enterica serovar enteritidis infection in virally immunosuppressed chickens. Clin. Diagn. Lab. Immunol. 10 (4), 670–679. Sigwart, D.F., Stocker, B.A., Clements, J.D., 1989. Effect of a purA mutation on efficacy of Salmonella live-vaccine vectors. Infect. Immun. 57 (6), 1858–1861. Stoicheva, M., Zlatev, S., Blagoeva, S., 1997. A study on some immunological parameters in infants with salmonellosis. Folia Med. (Plovdiv). 39, 17–23. van Deventer, S.J., 2000. Cytokine and cytokine receptor polymorphisms in infectious disease. Intensive Care Med. 26 (Suppl. 1), S98–S102. Villarreal, B., Mastroeni, P., de Hormaeche, R.D., Hormaeche, C.E., 1992. Proliferative and T-cell specific interleukin (IL-2/ IL-4) production responses in spleen cells from mice vaccinated with aroA live attenuated Salmonella vaccines. Microb. Pathogenesis 13, 305–315.
Salmonella enteritidis clearance and immune responses in chickens following Salmonella vaccination and challenge U. Babua,*, R.A. Dalloulb,c, M. Okamurab, H.S. Lillehojb, H. Xiec, R.B. Raybournea, D. Gainesa, R.A. Heckertc a
Center for Food Safety and Applied Nutrition, US Food and Drug Administration, HFS-326, 8301 Muirkirk Road, Laurel, MD 20708, USA b Animal Parasitic Diseases Laboratory, Animal and Natural Resources Institute, ARS, USDA, Building 1040 BARC-East, Beltsville, MD 20705, USA c Virginia–Maryland Regional College of Veterinary Medicine, University of Maryland, 8075 Greenmead Drive, College Park, MD 20742, USA Received 29 October 2003; received in revised form 6 May 2004; accepted 20 May 2004
Abstract Our previous work showed that the cell-mediated immunity (CMI) was enhanced by live Salmonella vaccine (LV). The objective of this study was to evaluate the impact of live and killed Salmonella vaccines on Salmonella enteritidis (SE) clearance and to determine if the clearance was mediated by cell-mediated and/or humoral immunity. Chickens were first immunized at 2 weeks of age followed by a booster dose at 4 weeks, challenged with live SE 2 weeks later (6-week-old) and tested for CMI, antibody response and SE clearance 1-week post SE-challenge (7-week-old). Spleen cell proliferation induced by SE-flagella and Concanavalin A (Con A) were significantly higher and SE shedding was significantly lower in the LV group. The splenic CD3 population was significantly lower and B cells were higher in the control group compared to all the SE-challenged groups (with and without vaccination). Serum antibody to SE-flagella and envelope were significantly higher in the KV group compared to all the other groups. These results suggest that LV protects against SE infection, probably by enhancing the CMI. # 2004 Elsevier B.V. All rights reserved. Keywords: Salmonella enteritidis; Vaccine; Chicken; Flagella; Lymphocyte proliferation
1. Introduction A significant proportion of human salmonellosis is known to be caused by consumption of raw or partially Abbreviations: CMI, cell-mediated immunity; DPI, days post immunization; DTH, delayed type hypersensitivity; HBSS, Hank’s balanced salt solution; h, hour(s); KV, killed vaccine; LV, live vaccine; min, minute; MNC, mononuclear cells; RT, room temperature; SE, Salmonella enteritidis; s, seconds * Corresponding author. Tel.: þ1 301 827 8515; fax: þ1 301 594 0517. E-mail address: [email protected] (U. Babu)
cooked eggs (Hope et al., 2002). On average, Americans consume 234 eggs/person/year. Salmonellaassociated food poisoning can result in significant financial burden on the health care system (Hasenson et al., 1992; Roberts and Sockett, 1994). Therefore, it is important to control egg contamination to reduce the worldwide salmonellosis problem. Besides good hygiene and animal husbandry practices, several methods have been currently employed to reduce Salmonella infection in poultry farms, such as the use of antibiotics, competitive exclusion by nonpathogenic bacteria, genetic selection of chicken strains for
0165-2427/$ – see front matter # 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2004.05.002
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U. Babu et al. / Veterinary Immunology and Immunopathology 101 (2004) 251–257
improved immune response, and development of Salmonella vaccines (Lillehoj et al., 2000). The efficacy of killed and live attenuated vaccine, and crude Salmonella immune lymphokines has been tested using chickens of different ages, various doses and routes of inoculation (Cooper et al., 1994; Hassan and Curtiss, 1990, 1994a, 1996). Generally, the immunopotentiating role of vaccines has been tested by their ability to stimulate antibody responses (Hassan and Curtiss, 1990, 1994a, 1996). Measurement of cell-mediated immunity (CMI) in vaccinated birds has been limited to the delayed type hypersensitivity (DTH) response to Salmonella typhimurium outer membrane proteins (Hassan and Curtiss, 1994b). Moreover, extensive mouse studies have shown the role for T cells, natural killer (NK) cells, macrophages, neutrophils and numerous cytokines in Salmonella resistance (as reviewed by Lillehoj and Okamura, 2003; Mastroeni et al., 2001; Mittrucker and Kaufmann, 2000). Our recently published study showed that a live vaccine (LV) was effective in enhancing the CMI of 18- and 32-week-old chickens as seen by the increased splenic lymphocyte proliferation to Salmonella enteritidis (SE) antigens and Con A (Babu et al., 2003). However, in that study we did not test if the increased CMI would influence SE clearance. Therefore, this study was conducted to evaluate the impact of live and killed vaccines on the ability of chickens to clear SE infection and to elucidate if the bacterial clearance was mediated by an enhanced immune response. Results from these studies showed that the live vaccine was more effective than the killed vaccine in clearing SE, which was accompanied by increased CMI. This may offer useful information for managing SE infection in chickens and shell egg contamination with SE.
2. Materials and methods 2.1. Birds, bacteria and vaccines Specific pathogen free White Leghorn chickens from Hyline International (Dallas Center, IA) were obtained as fertile eggs, hatched at the Animal and Natural Resources Institute facilities, Agricultural Research Service, US Department of Agriculture (Beltsville, MD). Chickens were kept in wire cages
and provided feed and water ad libitum with constant light source until use. One week before use, the birds were moved and housed in individual isolation units in a biosafety level 2 research facility at the College of Veterinary Medicine, University of Maryland (College Park, MD). Birds were provided with water and commercial antibiotic-free feed ad libitum. The experimental procedures and protocols were undertaken in accordance with the standards relating to the animal care and management guidelines of the University of Maryland. Salmonella enterica serovar Enteritidis phage type 4 strain 338 (Sheela et al., 2003) was diluted to 1 1010 CFU/ml in phosphate buffered saline (PBS) before inoculation. The live vaccine (Megan1Vac 1, Megan Health Inc., St. Louis, MO) was a Dcya Dcrp gene modified derivative of Salmonella typhimurium belonging to serogroup B (1, 4, 5, 12:i:1, 2). The killed vaccine (Poulvac1 SE, Ft. Dodge Animal Health Inc., Overland Park, KS) was an inactivated oil emulsion bacterin containing Salmonella enteritidis phage types 4, 8, and 13a belonging to the serogroup D1 (1, 9, 12:g, m:[1,7]). 2.2. Experimental design The objective of this study was to evaluate the impact of live and killed vaccines on SE clearance in young chickens. Twenty-four birds were assigned to four groups, namely the control (no vaccine, no challenge), challenge without immunization, the killed and the live vaccine groups with the SE-challenge. Birds in the vaccine groups were first immunized at 2 weeks of age and given a booster dose at 4 weeks of age. Two weeks later, these birds and those in the unvaccinated challenge group (6-week-old) were challenged with an oral infection of S. enteritidis (oral gavage, 1010 SE 338/bird). These birds were euthanized a week later (7-week-old) for immunological analyses and SE clearance. In this study, unvaccinated challenged birds served as control for the vaccinated and challenged birds. This offered us a way to determine the effect of infection alone or infection and immunization together on the immune response of young chickens. Furthermore, we could assess the efficacy of live and killed vaccine on SE clearance when compared to the unvaccinated challenge group.
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2.3. Flagella preparation from SE Flagella were prepared from Salmonella enteritidis strain 338 (SE 338) as described previously (Babu et al., 2003). Flagella antigen preparation primarily consisted of a single protein (approximately 54,000 Da) as confirmed by Coomassie Blue staining. 2.4. Isolation of splenic mononuclear cells Splenic mononuclear cells were isolated from fresh spleens as described previously (Babu et al., 2003). Cell concentration was adjusted to 8 106 cells/ml and viability was assessed by Trypan blue exclusion. 2.5. Mitogenic reactivity in vitro Splenic lymphocyte response to Con A- and SEflagella was assessed as described by Babu et al. (2003). Data are presented as counts per minute (cpm) from stimulated cultures after subtracting the cpm from non-stimulated cultures. 2.6. Antibodies and flow cytometric analysis Flow cytometric analysis of MNC was carried out as previously described (Sheela et al., 2003). MNC were incubated with R-phycoerythrin-conjugated anti-chicken CD3, and FITC-conjugated anti-chicken IgG (Southern Biotech, Birmingham, AL). Duplicate samples of MNC were stained with R-PE-anti-chicken CD4, and FITC-anti-chicken CD8 (Southern Biotech). R-PE- and FITC-conjugated isotype control samples were also included. Five thousand cells were analyzed using an EPICS Elite flow cytometer (Beckman/Coulter, Hialeah, FL). For gating purposes, the viable MNC population was determined by forward light scatter versus side light scatter analysis. 2.7. ELISA for serum antibodies Sera were tested for antibodies to two different Salmonella antigens, flagella and envelope. Flagella were prepared as described in the previous section. SE envelope was prepared by growing SE 338 in TSBYE broth overnight at 37 8C, the bacteria were washed twice with PBS and resuspended in 1 ml PBS for every 50 ml of original broth used. Cells were sonicated
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three times on ice for 1 min with 30 s of resting period after each sonication. Sonicated samples were centrifuged at 2700 rpm then the supernatant centrifuged at 15,000 rpm, each for 30 min at 4 8C. Flat bottom microtiter plates (Dynex Technologies, Chantilly, VA) were coated with 5.0 mg/well flagella Ag or 2.5 mg/well envelope Ag in 100 ml of 0.1 M carbonate buffer for 18 h at 4 8C. The plates were washed twice with PBS-T and blocking followed using 1% BSA– PBS for 30 min at RT. Plates were then flicked well and 100 ml of 1:100 diluted serum samples added to each well and allowed to incubate for 2 h at RT with continuous gentle shaking. The plates were washed 5 with PBS-T, 100 ml peroxidase-conjugated rabbit anti-chicken IgG mAb (dilution 1:2000; Sigma) were added to each well and allowed to incubate for 40 min at RT with shaking. The plates were washed again, 100 ml of TMB added as above, and the OD read at 450 nm using a plate reader. 2.8. Fecal shedding of SE This was carried out by the most probable number (MPN) estimation. Ceca from the six birds killed in each group were pooled and macerated. Isolation and enumeration of viable counts of SE from pools were made by direct plating of 10-fold dilutions of cecal contents on XLT Salmonella selective agar medium (Difco, Baltimore, MD) employing standard procedures (FDA Bacteriological Analytical Manual, 7th ed.). 2.9. Statistical analyses Data pertaining to proliferation and cell populations were compared between the control and the vaccine groups and among the vaccine groups by conducting one-way analysis of variance (ANOVA) followed by Student–Newman–Keuls test. These statistical analyses were done using Sigma Stat v2.01. Differences between the treatment groups were considered statistically significant at P < 0.05.
3. Results and discussion Chickens treated with LV prior to SE-challenge had reduced SE shedding (most probable number ¼ 20þ/g)
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Fig. 1. ConA (A) and SE-flagella (B) mediated spleen cell proliferation in chickens immunized with killed and live SE vaccines followed by live SE-challenge. Two-week-old chickens were immunized with killed and live SE vaccines (primary immunization), and were re-immunized (secondary immunization) 2 weeks later (age 4 weeks) and challenged 2 weeks later (age 6 weeks). SE infection was carried out with 1010 SE organisms. Birds were euthanized 1 week later (age 7 weeks) to assess splenic cell proliferation. Each bar represents mean S.E.M. (n ¼ 8). Bars without common letter differed (P < 0.05). SE: Salmonella enteritidis; KV: killed vaccine; LV: live vaccine.
compared to the unvaccinated birds or those that received the KV prior to challenge (most probable number ¼ 210þ/g for control and >1100þ/g for KV groups). These results are in agreement with previous reports of greater efficacy of live than the killed Salmonella vaccine in eliminating SE from chickens (Hassan and Curtiss, 1990; Mastroeni et al., 2001; Gast et al., 1993; Harrison et al., 1997). In addition to the increased SE clearance in the LV treated birds, spleen cells from these chickens responded significantly better to Con A and SE-flagella than those from the control and the KV groups Fig. 1A and B). These results are similar to our previous observations in older chickens treated with the same LV (Babu et al., 2003) and those from mouse studies in which attenuated SE and ST resulted in increased DTH and in vitro antigen-specific proliferation of spleen
cells, respectively (George et al., 1987; Villarreal et al., 1992). In contrast to the immunopotentiating effect of the live vaccine, we observed a strong negative effect of killed vaccine on the spleen cell proliferation to Con A. Other researchers have shown lower efficacy or immunosuppression by killed vaccine, compared to a more complete immune response elicited by live vaccine in chickens, mice and healthy human volunteers (George et al., 1987, 1989; Curtiss et al., 1993; Cooper, 1994; Harrison et al., 1997). Our results reestablished the importance of CMI in Salmonella clearance which was demonstrated previously in a mouse model (George et al., 1987; Mittrucker and Kaufmann, 2000; Mastroeni et al., 2001; Lalmanach and Lantier, 1999; Naiki et al., 1999). Infection of 6-week-old chickens with live virulent SE resulted in increased splenic CD3 and reduced B
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Table 1 Impact of live and killed Salmonella vaccine and SE-challenge on spleen T cell subsets and B cells of chickens Group
CD8
Control Control-challenge KV-challenge LV-challenge
36.6 43.2 38.9 40.3
CD4
5.5 3.5 6.1 0.52
15.6 16.5 21 18
CD3
3.7 2.5 5.8 4.7
49.2 56.3 58.2 56.2
B cells
6.1 b 1.1 a 5a 3.2 a
42.9 35.5 32.6 36.3
5.1 1.3 5.1 2.9
a b b b
Data are presented as mean S.D. (n ¼ 6 chickens/group). Two-week-old chickens were immunized with killed and live SE vaccines (primary immunization), and were re-immunized 2 weeks later (4-week-old secondary immunization) and challenged 2 weeks later (6-weekold). Challenge infection was carried out with 1010 SE organisms. Chickens were killed a week after challenge (7 weeks of age) and spleen cells were stained for CD4, CD8, CD3, or s-Igþ B cells. Cell subsets were determined by flow cytometry as described in Section 2. Means with different letters within the same column were different at P ¼ 0.05.
cell populations, with the highest CD3/B ratio in the KV group (Table 1). Although we observed increased CD3 cells in the infected groups, it was difficult to associate this increase with SE clearance due to lack of any significant changes in CD4þ or CD8þ cells (Mittrucker et al., 2002). Reduced B cells in Salmonella infected experimental calves and infants with salmonellosis has been reported previously (Liebler et al., 1994; Stoicheva et al., 1997). Importance of B cells in Salmonella infection has been established by reduced LD50 and impaired resistance to Salmonella typhimuriun in B cell deficient mice and by decreased SE clearance in young chickens treated with testosterone propionate which selectively depletes B cells (Mastroeni et al., 2000; Arnold and Holt, 1995). In our study, although B cell depletion was similar in all the challenged birds, SE clearance was significantly higher in the LV group. This suggests that LV produces factors that can possibly overcome the B cell deficit and clear the SE infection in chickens. This speculation is based on live Salmonella typhimurium induced increase in IFN-g, IL-12, and IL-2 (John et al., 2002; Harrison et al., 1997), which are known to play a role in Salmonella clearance (Dybing et al., 1999; van Deventer, 2000). In contrast to its inhibitory effect on CMI and SE clearance, KV induced highest SE-specific antibody (Fig. 2). It is not clear if this increased antibody response is associated with an increased T to B cell ratio in the KV group (Brieva et al., 1983). Our results suggest that the antibody against SE does not protect the chickens against the SE infection. There is some controversy about the involvement of humoral immune response in protection against Salmonella infection, with some studies showing a positive cor-
relation between antibody response and Salmonella clearance and others showing no effect (Hassan and Curtiss, 1990, 1994b; Begg et al., 1990; Sigwart et al., 1989). Overall, this is one of the few studies showing that oral vaccination with LV enhanced CMI with increased SE clearance in chickens. In contrast to the effects of LV, KV enhanced the humoral immune response, significantly inhibited the CMI and increased the bacterial load in young chickens, thus indicating the importance of CMI in SE clearance. Our results suggest that vaccination with attenuated live Salmonella could be an effective method for
Fig. 2. Antibody response in challenged birds following secondary Salmonella immunization. Sera were tested for Abs against flagella and envelope antigens. Each bar represents the mean of three to six birds. Two-week-old chickens were immunized with killed and live SE vaccines (primary immunization), and were re-immunized (secondary immunization) 2 weeks later (age 4 weeks) and challenged 2 weeks later (age 6 weeks). SE infection was carried out with 1010 SE organisms. Each bar represents mean S.E.M. (n ¼ 6). Bars without common letter differed (P < 0.05). SE: Salmonella enteritidis; KV: killed vaccine; LV: live vaccine.
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controlling Salmonella enteritidis infection in young chickens.
Acknowledgements This work was supported by the grant from USDA National Food Safety Initiative Grant 00-51110-9739. We are grateful to S. Elankumaran, R. Sheela, Elmer Bigley and Terry Gaither for their excellent and valuable support in completion of this project. We would like to thank Sandra Kelly-Aehle from Megan Health Inc., for providing the live attenuated vaccine and Jerry D. Maiers, Senior Poultry Technical Services Veterinarian, Fort Dodge Animal Health, for providing the killed vaccine.
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