"BRUCELLOSIS VACCINES FOR LIVESTOCK"

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1000663
• Grant No.: 2013-67015-21187
• Project No.: FLAW-2013-01165
• Proposal No.: 2013-01165
• Program Code: A1221
• Project Start Date: Sep 1, 2013
• Project End Date: Aug 31, 2018
• Grant Year: 2013

Project Director
Pascual, D. W.

Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611

Performing Department
Infectious Diseases &Pathology

Non Technical Summary
Brucella abortus remains a threat to the health and well-being of livestock in states bordering the Greater Yellowstone Area (GYA). Cohabitation of infected wildlife with cattle has jeopardized the brucellosis-free status of Montana, Idaho, and Wyoming and caused a recent outbreak in Texas. Consequently, infected wildlife will continue to be problematic unless some intervention or vaccination program is initiated to allow adequate management of this disease. The current livestock RB51 vaccine has failed to confer protection of livestock in the affected areas. Thus, if an improved vaccine can be developed, this should lessen the impact of infected wildlife. To forward such effort, we have developed a next generation double mutant vaccine that shows 100% efficacy in a mouse model and, in some cases, provides sterile immunity against B. abortus challenge. The proposed studies will test this vaccine in relevant animals, calves, to determine if this vaccine shows improved efficacy over conventional RB51 vaccine. Studies will also determine whether a single or two-dose is required for protection. Additional modifications to the vaccine will be made to define a suitable marker gene to enable differentiating infected from vaccinated animals (DIVA) since the B. abortus LPS remains. Finally, studies will assess the induced T cell responses to obtain correlates of protection. From these studies, an improved brucellosis vaccine will be developed to enhance long-range improvement in and sustainability of US agriculture and food systems.

Animal Health Component

100%

Research Effort Categories

Basic

35%

Applied

65%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3310	1090	60%
311	3310	1100	15%
311	3410	1090	10%
311	3410	1100	15%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3310 - Beef cattle, live animal; 3410 - Dairy cattle, live animal;

Field Of Science
1090 - Immunology; 1100 - Bacteriology;

Keywords

brucellosis

vaccine

Goals / Objectives
Goal 1: Determine the efficacy of subcutaneous ΔnorD ΔznuA B. abortus-lacZ vaccine in calves against S19 challenge. Goal 2: Determine correlates of protection conferred by ΔnorD ΔznuA B. abortus-lacZ-vaccinated animals prior to and after S19 challenge. Goal 3: Develop a suitable marker gene to facilitate differentiating infected from vaccinated animal (DIVA)

Project Methods
In Vivo Colonization Studies of Calves with ΔnorD ΔznuA B. abortus-lacZ Vaccine This set of studies will be performed in two phases: the first will assess the level of colonization of the vaccine in calves, followed by the second phase to evaluate the efficacy of this vaccine against S19 challenge. Vaccine production. ΔnorD ΔznuA B. abortus-lacZ will be grown overnight in Brucella Broth (BB) at 37°C. The vaccine strains will be pelleted, washed twice in sterile phosphate-buffered saline (sPBS), and diluted to 1×1010 cells/2.0 ml in sPBS. The actual viable inoculum CFU will be confirmed by serial dilution tests on Bacto Potato Infusion Agar (PIA). Immunization. Fifteen beef calves approximately six months of age will be tested for brucellosis (Card determination) and selected for not being previously immunized with RB51. On day 0, all calves will be vaccinated subcutaneously with 1×1010 CFUs ΔnorD ΔznuA B. abortus-lacZ administered to the neck as done for RB51. Colony counts will be performed to confirm the vaccine dosage administered. On days 3, 7, 14, 21, and 28, three calves/day will be euthanized for their peripheral blood, spleens, and primary and secondary lymph nodes, and these tissues will be isolated, weighed, and homogenized in sPBS to assess extent of colonization; colony counts then will be performed in triplicate for each heifer. Duplicate CFU determinations will be performed to determine ß-galactosidase activity. For each day, CFU counts will be averaged + SEM. Since we have previously shown that the vaccine reduced three to four logs within 4-6 wks in the mouse, these data should be informative in providing when the vaccine is mostly cleared. We can easily amend the sampling time points as we learn the clearance rates. Vaccine Efficacy Study in Calves - Two-dose Study Immunizations. A power analysis was performed, and it was determined at least 8 calves/group were needed for P < 0.05. Twenty-four beef calves ~8 months of age, will be split into three groups: eight heifers will be immunized with 1x1010 CFUs of ΔnorD ΔznuA B. abortus-lacZ; eight heifers will be immunized with 1x1010 CFUs RB51, and eight heifers will serve as a PBS control group. Four weeks after primary immunization, the ΔnorD ΔznuA B. abortus-lacZ-vaccinated heifers will each receive a booster immunization with the same dose using the same route of immunization. Control heifers will receive sPBS, and RB51-vaccinated animals will be left alone. Challenge studies. To ensure the vaccines have cleared from tissues, a punch biopsy of two superficial cervical lymph nodes will be done 4 wks after the last immunization to assess vaccine levels in these lymph nodes. Since ΔnorD ΔznuA B. abortus-lacZ has the marker gene encoding the enzyme, ß-galactosidase, growth in the presence of substrate will turn vaccine colonies blue; thus, we should be able to readily detect the vaccine if it is present in the tissues. Since RB51 is a "rough" mutant, we can distinguish this strain by its morphology. If vaccine brucellae are still detectable, the challenge will be postponed by an additional 3 wks, and lymph nodes will be reevaluated. Once the absence of vaccine growth can be demonstrated, all heifers will be challenged with S19. Briefly, B. abortus S19 will be grown overnight in BB at 37°C. The vaccinated and naive groups will be challenged via the conjunctival sac with 1x109 CFUs in 100 µl of B. abortus S19, as previously described. Colony counts will be performed to confirm vaccine dosage administered. The individual heifers will be euthanized 28 days after challenge, and peripheral blood and their spleens and lymph nodes (primary and secondary) will be isolated, weighed, and homogenized in sPBS to assess extent of colonization; colony counts then will be performed in triplicate for each heifer. Duplicate CFU determinations will be done to ascertain ß-galactosidase activity from the vaccinated animals to ensure the vaccine has been cleared. For each group, CFU counts will be averaged + SEM. Extent of colonization will be compared between immunized and PBS-dosed groups. The latter group should represent unprotected heifers and have maximal CFU counts. PCR will also be performed to confirm these CFU levels represent the challenge strain and not the vaccine strain. From these studies, we will learn the level of protection conferred by ΔnorD ΔznuA B. abortus-lacZ relative to RB51 and will provide data to warrant to pursue a future abortion trial. T Cell Evaluations. Peripheral blood mononuclear cells and lymph node punch biopsies will be our source of immune lymphocytes before and after S19 challenge. CD4+ and CD8+ T cell responses will be evaluated using mononuclear cell cultures. Lymphocytes will be cultured either without or with varying dilutions (1x107 to 1x109 CFUs/well) of heat-killed RB51 (brucellae will be tested for viability to ensure it is completely dead) or an irrelevant antigen, 1.0 mg OVA for 3 - 5 days. During the last 16 hrs of culture, cells will be pulsed with 3H-thymidine to examine the level of incorporation in response to Brucella antigen. From these studies, we expect to observe enhanced responsiveness by the CD4+ T cells from immunized calves when compared to CD4+ T cells from PBS-dosed bovine lymphocytes. The following studies will determine whether the CD4+ T cells exhibit a Th1, Th2, or Th17 cell bias. To distinguish between these Th cell subsets, cytokine-specific ELISA/ELISPOT assays will be used: the Th1 cell cytokines, IL-2, TNF-α, and IFN-γ; Th2 cell cytokines: IL-4 and IL-10; and Th17 cell cytokine: IL-17. From these cytokine analyses, we will learn whether enhanced Th1 and Th17 cells will be induced relative to Th2 cells. We anticipate increases in IFN-γ and IL-17 will account for the efficacy by ΔnorD ΔznuA B. abortus vaccine. Construction of new DIVA-bearing vaccines by chromosomal insertion. To construct our ΔnorD ΔznuA B. abortus vaccine, a lacZ expression cassette will be introduced into the vaccine's chromosome. The cfaB or Hc/A into ΔnorD ΔznuA B. abortus by replacing the lacZ cassette with either cfaB or Hc/A expression cassettes. The genes for cfaB and Hc/A will be codon-optimized for B. abortus. The synthesized genes will be under the control of suitable promoters, as done for lacZ. Newly constructed suicide plasmids will be transferred into ΔnorD ΔznuA B. abortus to replace lacZ. The recombinant proteins produced by ΔnorD ΔznuA B. abortus strains will be confirmed by Western blot to ensure expression of these DIVA proteins.

Progress 09/01/13 to 08/31/18

Outputs
Target Audience:Brucella abortus remains a threat to the health and well-being of livestock in states bordering the Greater Yellowstone Area (GYA). Cohabitation of infected wildlife with cattle has jeopardized the brucellosis-free status of Montana, Idaho, and Wyoming and caused a recent outbreak in Texas. Consequently, infected wildlife will continue to be problematic unless some intervention or a vaccination program is initiated to allow adequate management of this disease. The current livestock RB51 vaccine has failed to confer protection of livestock in the affected areas. Thus, if an improved vaccine can be developed, this should lessen the impact from infected wildlife. To forward such effort, we have developed a next generation double mutant vaccine that shows 100% efficacy in a mouse model and, in some cases, provides sterile immunity against B. abortus challenge. The proposed studies will test this vaccine in relevant animals, calves, to determine if this vaccine shows improved efficacy. Studies will also determine whether a single or two-dose is required for protection. Additional modifications to the vaccine will be made to define a suitable marker gene to enable differentiating infected from vaccinated animals (DIVA) since the B. abortus LPS remains. Finally, studies will assess the induced T cell responses to obtain correlates of protection. From these studies, an improved brucellosis vaccine will be developed to enhance long-range improvement in and sustainability of US agriculture and food systems. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This work has offered training and professional development for my graduate student, Dr. Zakia Goodwin, who completed her Master's thesis in August 2015. This project gave her an excellent opportunity for her to learn how to conduct scientific research. She did not have such opportunity during her D.V.M. training. This work has also provided her a chance to learn about brucellosis vaccine responses in calves and how these compare to RB51-vaccinated animals. She is currently pursuing a Ph.D. in my laboratory as of August 2016. How have the results been disseminated to communities of interest?We have reported our results as a M.S. thesis by my former graduate student, Dr. Zakia Goodwin. Some of the work has been reported in three publications in infectious disease and vaccine-related journals, and presented 3 papers at national and international conferences. We currently have one review article and one book chapter on brucellosis vaccines in press for publication. What do you plan to do during the next reporting period to accomplish the goals?We are developing a new vaccine strain that is expected to be more potent in livestock. We suspect the original strain was eliminated too quickly from the calves to elicit a protective immune response.

Impacts
What was accomplished under these goals? Goals 1 & 2: The vaccine trial was conducted where calves were s.c. vaccinated once or twice with 1x1010 CFUs of the double-mutant vaccine, and the results were compared to calves dosed only with sterile PBS. Vaccination with the double-mutant vaccine showed enhanced antigen-specific bovine T cell proliferative responses to heat-killed (HKRB51) relative to media stimulation. Calves vaccinated once or twice showed significant proliferation by their peripheral blood T cells (p <0.05). Evaluation of IFN-γ and IL-17 responses from these same calves was measured using cytokine-specific ELISAs. Cell culture supernatants from HKRB51-restimulated PBMCs showed significant increases in IFN-γ and IL-17 (p < 0.05). IL-10 was also induced by reactivated PBMCs from calves vaccinated once, but was reduced by reactivated PBMCs from twice-vaccinated calves. All calves were challenged with B. abortus S19 via the conjunctival route 4 weeks after the last dose of vaccine. Calves were euthanized 2 weeks after challenge, and regional and distal lymph nodes, spleens, and ovaries were tested for brucellae persistence. While the twice vaccinated calves showed a trend for reduced tissue colonization, neither vaccinated group was significantly different from PBS-dosed calves. The IFN-γ production failed to correlate to reduction in brucellae colonization of the primary lymph nodes, e.g., parotid lymph nodes, submandibular lymph nodes, and retropharyngeal lymph nodes, after B. abortus S19 challenge. Goal 3: We have completed the development of the modified double-mutant vaccine with the new DIVA gene for mCherry to replace lacZ. We have recently developed an improved expression system for Brucella strains. This has considerably enhanced the immunogenicityof DIVA gene products.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Goodwin ZI, Pascual DW. 2016. Brucellosis vaccines for livestock. Vet Immunol Immunopathol. 181:51-58. Clapp, B., Yang, X., Thornburg, T., Walters, N., and Pascual, D.W. 2016. Nasal vaccination stimulates CD8+ T cells for potent protection against mucosal Brucella melitensis challenge. Immunol. Cell Biol. 94:496-508. Yang, X., Clapp, B., Thornburg, T., Hoffman, C., and Pascual, D.W. 2016. Vaccination with a DnorD DznuA Brucella abortus mutant confers potent protection against virulent challenge. Vaccine 34:5290-5297. Pascual, D.W., Yang, X., Wang, H., Goodwin, Z., Hoffman, C., and Clapp, B. 2018. Alternative strategies for vaccination to brucellosis. Microbes & Infect. 20: (In press).
• Type: Book Chapters Status: Awaiting Publication Year Published: 2019 Citation: Pascual, D.W. 2018. Mucosal approaches for systemic immunity to anthrax, brucellosis, and plague. Mucosal Vaccines, 2nd ed., H. Kiyono and D.W. Pascual, eds., Elsevier, Inc., S�o Paulo, Brazil, Chap. 28 (in press).
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Pascual, D.W., Yang, G., Wang, H., Diaz-Goodman, Z., Hoffman, C., and Clapp, B. Alternative immune mechanisms for protection against brucellosis. 98th Annual Conference of Research Workers in Animal Diseases, Chicago, IL, Dec. 3-5, 2017. Clapp, B., Guan, Y., Artiaga, B.L., Hoffman, C., Yang, X., Driver, J.P., and Pascual, D.W. Development of a swine vaccination model for human brucellosis. 70th Annual Brucellosis Research Conference, Chicago, IL, Dec. 2-3, 2017.

Progress 09/01/15 to 08/31/16

Outputs
Target Audience:Brucella abortus remains a threat to the health and well-being of livestock in states bordering the Greater Yellowstone Area (GYA). Cohabitation of infected wildlife with cattle has jeopardized the brucellosis-free status of Montana, Idaho, and Wyoming and caused a recent outbreak in Texas. Consequently, infected wildlife will continue to be problematic unless some intervention or a vaccination program is initiated to allow adequate management of this disease. The current livestock RB51 vaccine has failed to confer protection of livestock in the affected areas. Thus, if an improved vaccine can be developed, this should lessen the impact from infected wildlife. To forward such effort, we have developed a next generation double mutant vaccine that shows 100% efficacy in a mouse model and, in some cases, provides sterile immunity against B. abortus challenge. The proposed studies will test this vaccine in relevant animals, calves, to determine if this vaccine shows improved efficacy. Studies will also determine whether a single or two-dose is required for protection. Additional modifications to the vaccine will be made to define a suitable marker gene to enable differentiating infected from vaccinated animals (DIVA) since the B. abortus LPS remains. Finally, studies will assess the induced T cell responses to obtain correlates of protection. From these studies, an improved brucellosis vaccine will be developed to enhance long-range improvement in and sustainability of US agriculture and food systems. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This work has offered training and professional development for my graduate student, Dr. Zakia Goodwin, who completed her master's thesis in August 2015. This project gave her an excellent opportunity for her to learn how to conduct scientific research. She did not have such opportunity during her D.V.M. training. This work has also provided her a chance to learn about brucellosis vaccine responses in calves and how these compare to RB51-vaccinated animals. She will be pursuing a Ph.D. in my laboratory beginning in August 2016. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?We plan to complete the S19 challenge analysis to determine if the calves were sufficiently vaccinated. We are striving to learn what are the correlates of immune protection to brucellosis in calves.

Impacts
What was accomplished under these goals? Goals 1 & 2: As described in the previous report, we have initiated the vaccine trial where calves were s.c. vaccinated once or twice with 1x1010 CFUs of the double-mutant vaccine, and the results were compared to calves dosed only with sterile PBS. Vaccination with the double-mutant vaccine showed enhanced antigen-specific bovine T cell proliferative responses to heat-killed (HKRB51) relative to media stimulation Calves vaccinated once or twice showed significant proliferation by their peripheral blood T cells (p <0.05). Evaluation of IFN-ã and IL-17 responses from these same calves was measured by using cytokine-specific ELISAs. Cell culture supernatants from HKRB51-restimulated PBMCs showed significant increases in IFN-γ and IL-17 (p < 0.05). IL-10 was also induced by reactivated PBMCs from calves vaccinated once, but was reduced by reactivated PBMCs from twice-vaccinated calves. All calves were challenged with B. abortus S19 via the conjunctival route 4 weeks after the last dose of vaccine. Calves were euthanized 2 weeks after challenge, and regional and distal lymph nodes, spleens, and ovaries were tested for brucellae persistence. While the twice vaccinated calves showed a trend for reduced tissue colonization, neither vaccinated group was significantly different from PBS-dosed calves. Current work is analyzing data to determine if cytokine profiles correlate to reduced brucellae colonization in the affected calves. Goal 3: We have completed the development of the modified double-mutant vaccine with the new DIVA gene for mCherry to replace lacZ. We are currently evaluating host antibody responses to mCherry for this new construct.

Publications

• Type: Journal Articles Status: Awaiting Publication Year Published: 2016 Citation: Goodwin, Z.I. and Pascual, D.W. 2016. Brucellosis vaccines for livestock. Vet. Immunol. Immunopath. (In press). S0165-2427(16)30036-8. doi: 10.1016/j.vetimm.2016.03.011
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Clapp, B., Yang, X., Thornburg, T., Walters, N., and Pascual, D.W. 2016. Nasal vaccination stimulates CD8+ T cells for potent protection against mucosal Brucella melitensis challenge. Immunol. Cell Biol. 94:496-508.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Pascual, D.W., Wang, H., Yang, X., and Clapp, B. Varied innate cell responses in the lungs following live vector vaccination or wild-type Brucella infection. The 14th Awaji International Forum on Infection and Immunity, Awaji Island, Japan, Sept. 8-11, 2015.
• Type: Theses/Dissertations Status: Published Year Published: 2015 Citation: Zakia I. Goodwin-Diaz, Masters student (2013-2015), completed thesis: Immunogenicty and Protective Response of a Live Attenuated Double Mutant Vaccine Against Brucella abortus in Cattle, in August, 2015, ID&P, Univ. of Florida, Gainesville, FL.

Progress 09/01/14 to 08/31/15

Outputs
Target Audience:Brucella abortus remains a threat to the health and well-being of livestock in states bordering the Greater Yellowstone Area (GYA). Cohabitation of infected wildlife with cattle has jeopardized the brucellosis-free status of Montana, Idaho, and Wyoming and caused an outbreak in Texas. Consequently, infected wildlife will continue to be problematic unless some intervention or vaccination program is initiated to allow adequate management of this disease. The current livestock RB51 vaccine has failed to confer protection of livestock in the affected areas. Thus, if an improved vaccine can be developed, this should lessen the impact of infected wildlife. To forward such effort, we have developed a next generation double mutant vaccine that shows 100% efficacy in a mouse model and, in some cases, provides sterile immunity against B. abortus challenge. The proposed studies will test this vaccine in relevant animals, calves, to determine if this vaccine shows improved efficacy over conventional RB51 vaccine. Studies will also determine whether a single or two-dose is required for protection. Additional modifications to the vaccine will be made to define a suitable marker gene to enable differentiating infected from vaccinated animals (DIVA) since the B. abortus LPS remains. Finally, studies will assess the induced T cell responses to obtain correlates of protection. From these studies, an improved brucellosis vaccine will be developed to enhance long-range improvement in and sustainability of US agriculture and food systems. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This work has offered training and professional development for my graduate student, Dr. Zakia Goodwin, who is working to complete her master's thesis. This project has provided an excellent opportunity for her to learn how to conduct scientific research. She did not have such opportunity during her D.V.M. training. This work has also provided her a chance to learn about brucellosis vaccine responses in calves and how these compare to RB51-vaccinated animals. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?We plan to complete the S19 challenge studies to learn if the double-mutant vaccine will be efficacious against tissue brucellae colonization in calves.

Impacts
What was accomplished under these goals? Goals 1 & 2: As described in the previous report, studies were conducted to analyze the clearance rate of the double-mutant vaccine as well as to ascertain the immunogenicity of the vaccine at the latter time points of in vivo clearance analysis. During this past year, we have initiated the vaccine trial and have completed the immune analyses for the calves that will be challenged. Vaccination with the double-mutant vaccine enhances antigen-specific bovine T cell proliferative responses. PBMCs were isolated from calves at 0, 4, and 8 weeks post-vaccination. Cells were cultured for 72 hrs restimulated with 108 CFUs of heat-killed RB51 (HKRB51) or left unstimulated as a negative stimulation control. Cells were pulsed for the last 18 hrs of culture with 0.5 µCi 3H-thymidine to measure cell proliferation. Calves dosed once or twice showed significant proliferation (p <0.05). Evaluation of IFN-γ and IL-17 responses from these same calves given one or two doses of double-mutant vaccine was accomplished by using cytokine-specific ELISAs. Cell culture supernatants from HKRB51-restimulated PBMCs showed significant increases in IFN-γ and IL-17 (p < 0.05). IL-10 was also induced after a single dose, but was reduced in twice-vaccinated calves. Goal 3: We have completed the development of the modified double-mutant vaccine with the new DIVA gene for mCherry to replace lacZ. We are currently evaluating host antibody responses to mCherry for this new construct.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Pascual, D.W., Clapp, B., Wang, H., Diaz-Goodwin, Z., Hoffman, C., and Yang, X. Mucosal vaccines for brucellosis. Keystone Symposium: Immunity to Veterinary Pathogens: Informing Vaccine Development, January 20-25, 2015, Keystone, CO.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Pascual, D.W., Wang, H., Yang, X., and Clapp, B. Nasal vaccination induces innate lymphoid cells (ILC1s) and NK cells to initiate the lung IFN-g cascade that supports adaptive T cells responses to Brucella. 17th Intern. Cong. Mucosal Immunol., Berlin, Germany, July 14-18, 2015, OR.76.
• Type: Journal Articles Status: Submitted Year Published: 2015 Citation: Goodwin, Z.I. and Pascual, D.W. Brucellosis vaccines for livestock. Vet. Immunol. Immunopath.

Progress 09/01/13 to 08/31/14

Outputs
Target Audience: Brucella abortus remains a threat to the health and well-being of livestock in states bordering the Greater Yellowstone Area (GYA). Cohabitation of infected wildlife with cattle has jeopardized the brucellosis-free status of Montana, Idaho, and Wyoming and caused a recent outbreak in Texas. Consequently, infected wildlife will continue to be problematic unless some intervention or vaccination program is initiated to allow adequate management of this disease. The current livestock RB51 vaccine has failed to confer protection of livestock in the affected areas. Thus, if an improved vaccine can be developed, this should lessen the impact of infected wildlife. To forward such effort, we have developed a next generation double mutant vaccine that shows 100% efficacy in a mouse model and, in some cases, provides sterile immunity against B. abortus challenge. The proposed studies will test this vaccine in relevant animals, calves, to determine if this vaccine shows improved efficacy over conventional RB51 vaccine. Studies will also determine whether a single or two-dose is required for protection. Additional modifications to the vaccine will be made to define a suitable marker gene to enable differentiating infected from vaccinated animals (DIVA) since the B. abortus LPS remains. Finally, studies will assess the induced T cell responses to obtain correlates of protection. From these studies, an improved brucellosis vaccine will be developed to enhance long-range improvement in and sustainability of US agriculture and food systems. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This work has offered training and professional development for my graduate student to learn how to conduct scientific research. This is a unique opportunity to learn brucellosis vaccine responses in calves and compare how these differ from RB51-vaccinated animals. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? We plan to undertake the vaccination of calves with the double-mutant and S19 challenge studies to learn correlates of protection to B. abortus.

Impacts
What was accomplished under these goals? For Objective 1, the first phase of work was designed to determine the clearance of the double-mutant brucellosis vaccine from calves. Fifteen beef calves (9 months of age) were obtained from a local herd. Animals were brucellosis-negative and not previously immunized with RB51. Calves were randomly assigned to five groups (n=3 animals/group) for a single subcutaneous vaccination with 1010 CFUs of double-mutant vaccine. On days 1-3 post-vaccination, nasal, oral, and fecal swabs were collected to determine Brucella presence in these mucosal sites. Animals (3/time point) were euthanized on days 3, 7, 14, 21, and 28 post-vaccination and peripheral blood, spleens, ovaries, ileum, and primary and secondary lymph nodes. The mutant strain could not be recovered from any of the examined tissue samples at any of the time points tested nor in any of the mucosal or blood samples. For Objective 2, studies are focused on understanding bovine immune responses to the double-mutant vaccine. T cell proliferation and IFN-γ responses were measured using peripheral blood mononuclear cells (PBMCs) at 2 weeks post-vaccination on the remaining 9 calves. For proliferation assay, PBMCs (5×105) were cultured in the presence of 2x108 CFUs of heat-killed RB51 (HKRB51) or no additives (unstimulated media control). Since RB51 lacks its LPS, the induced T cell responses are directed against Brucella proteins. The cells were cultured for 4 days and examined for 3H-thymidine uptake. Lymphocytes from all vaccinated calves showed greater proliferative responses between 3.2- to 17.4-fold to HKRB51 compared to unstimulated control cells. For IFN-γ production, lymphocytes from the same vaccinated calves were cultured in medium alone or with 109 CFUs/ml of HKRB51 for 3 days. The supernatants were evaluated using a bovine-specific capture ELISA to quantify IFN-γ levels. Upon restimulation with HKRB51, the PBMCs from vaccinated animals (7 of 9 calves) showed significantly greater levels of IFN-γ relative medium-stimulated cells (P < 0.05). For Objective 3, we have secured permission from USDA-APHIS to generate new DIVA insertions into our double-mutant vaccine.

Publications