Progress 05/01/08 to 04/30/13
Outputs
Progress Report Objectives (from AD-416): The objective of this collaborative agreement is to gain a better understanding of specific immune response to Foot-and-Mouth Disease Virus (FMDV) infection and vaccination and to identify the genetic basis of animals with high and low responder phenotypes. The objectives are: 1. to describe the early steps of the specific immune response against FMDV infection, 2. to describe the immune response elicited by inactivated FMDV vaccination, and 3. to determine the heritability of the response to FMDV vaccination in cattle populations. Approach (from AD-416): 1. The early steps of specific immune response against FMDV infection will be done through infection by aerosol inoculation delivery route and subsequent virus detection of respiratory tissues through immunostaining and real-time RT-PCR will be done. Replication sites will be correlated to anti-FMDV antibody-producing cells and studied. 2. The immune response elicited by inactivated FMDV vaccination will be studied. Cells responsible for antibody production will be determined through ELISpot assay and analyzed for protective response elicted by inactivated FMDV vaccines. These cells will genotyped with the aim of correlating host genotype with host resistance/recovery after experimental infection or induction of protection post vaccination. Results obtained from in vivo experiments will then be compared to in vitro responses. 3. The heritability of response to FMDV vaccination will be conducted studies of vaccinated bovine populations in Argentina with known pedigree. Estimates of heritability will be calculated to assess host genetic influence to FMDV vaccination response. These estimates will be used to assess merit of further efforts to identify bovine strains purported to demonstrate enhanced levels of innate resistance to FMDV. This will determine if selection for host resistance is possible and if significant gentetic differentiation is possible to identify. The aim of this project is to better understand the specific immune response to foot and mouth disease virus (FDMV) infection and vaccination and to identify the genetic basis of animals with high and low responder phenotypes. The project focuses on two main objectives: 1) To describe the early steps of the specific immune response against the FMDV infection in na�ve and vaccinated cattle and; 2) To determine the heritability of the response to FMDV vaccination in cattle populations. Studies were completed for objective 1 inclusive of (1) the assaying the seroneutralizing activity of the FMDV-specific antibodies found at systemic level, (2) conducted studies of viremia in the serum samples of the vaccinated-infected animals, and (3) the establishment of a FMDV- specific B-cell memory assay. Results of these studies indicate that the FMDV-specific antibody secreting cells (ASC) could be detected in lymph nodes from respiratory tract since 7 days after intramuscular administration of a high-payload FMDV-inactivated oil vaccine. Also, presence of vaccine-induced FMDV- specific memory B-cells was evidenced in these tissues after oronasal challenge performed in these vaccinated cattle at 30 days post vaccination (dpv). In objective 2, the heterogeneity in the antibody response of cattle primo-vaccinated against FMD were quantified and its association with the genetic background in Holstein and Jersey sires was studied. A total of 377 FMDV seronegative calves, 122 and 255 calves from 16 and 15 Holstein and Jersey sires, respectively, were included in the study. Samples were taken the day prior to primo-vaccination and 45 dpv. Animals received commercial tetravalent FMD single-emulsion oil vaccines formulated with inactivated FMDV. Total FMDV-specific antibody responses were studied against 3 viral strains included in the vaccine and antibody titers were determined. Three linear hierarchical mixed regression models, one foreach strain, were formulated based on the dependent variables. Results of these studies indicate that breed may have a significant effect in the humoral immune response elicited after immunization with high-potency commercial FMD vaccines. Progeny of sires of Jersey breed developed a humoral response 45days post-primo vaccination which was significantly lower than Holstein sires� offspring. No differences were found for calves derived from sires within the same breed. Analyses were carried out independently for three vaccine strains included in the formulation and results were similar in all cases. The significant accomplishments include; 1. The development of FMDV- specific ASC and interferon gamma � ELISpot assays to characterize local adaptive humoral responses induced in the bovine respiratory tract after FMDV infection. No previous references to these assays were described in the literature. 2. A detailed description of actual local responses elicited at early times after experimental oronasal FMDV-infection in na�ve and vaccinated cattle using both techniques mentioned above. 3. A description of the presence of memory B-cells in the respiratory tract lymph nodes after intramuscular immunization with high-potency commercial FMD vaccines. 4. Results indicating that no differences were detected in the systemic antibody response induced at 45 dpv by high-potency commercial FMD vaccines among calves derived from sires of the same breed. The mean antibody titers differed only for calves from different breeds (Jersey vs. Holstein). No technologies have been transferred during this period. Publications for FY 2013 include: Early adaptive immune responses in the respiratory tract of foot and mouth disease-infected cattle. J. Pega, D. Bucafusco, S. Di Giacomo, JM. Schammas, D. Malacari, A. Capozzo, J. Arzt, C. P�rez-Beascoechea, E. Maradei, LL. Rodr�guez, MV. Borca, M. P�rez-Filgueira. 2013, J Virol 87: 2489-95. Heterogeneity in the antibody response to foot-and-mouth disease primo vaccinated calves. S. Di Giacomo (*); B. Brito (*); A. M. Perez; D. Bucafusco; J. Pega; L. Rodr�guez; M. Borca and M. P�rez-Filgueira. 2013, Transboundary and Emerging Diseases (in press). 2012 Meeting of the European Commission for the Control of Foot-and- Mouth Disease (EuFMD) �Appliance of science in the progressive control of FMD� held in Jerez de la Frontera, Spain, Octuber 29 -31, 2012 (�Adaptative immune responses in the respiratory tract of FMD-vaccinated cattle after oronasal infection� J. Pega, S. Di Giacomo, D. Bucafusco, J. Schammas, D. Malacari, G. Stafforini, A. Capozzo, LL. Rodr�guez, MV. Borca, M. P�rez-Filgueira).
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Progress 10/01/11 to 09/30/12
Outputs
Progress Report Objectives (from AD-416): The objective of this collaborative agreement is to gain a better understanding of specific immune response to Foot-and-Mouth Disease Virus (FMDV) infection and vaccination and to identify the genetic basis of animals with high and low responder phenotypes. The objectives are: 1. to describe the early steps of the specific immune response against FMDV infection, 2. to describe the immune response elicited by inactivated FMDV vaccination, and 3. to determine the heritability of the response to FMDV vaccination in cattle populations. Approach (from AD-416): 1. The early steps of specific immune response against FMDV infection will be done through infection by aerosol inoculation delivery route and subsequent virus detection of respiratory tissues through immunostaining and real-time RT-PCR will be done. Replication sites will be correlated to anti-FMDV antibody-producing cells and studied. 2. The immune response elicited by inactivated FMDV vaccination will be studied. Cells responsible for antibody production will be determined through ELISpot assay and analyzed for protective response elicted by inactivated FMDV vaccines. These cells will genotyped with the aim of correlating host genotype with host resistance/recovery after experimental infection or induction of protection post vaccination. Results obtained from in vivo experiments will then be compared to in vitro responses. 3. The heritability of response to FMDV vaccination will be conducted studies of vaccinated bovine populations in Argentina with known pedigree. Estimates of heritability will be calculated to assess host genetic influence to FMDV vaccination response. These estimates will be used to assess merit of further efforts to identify bovine strains purported to demonstrate enhanced levels of innate resistance to FMDV. This will determine if selection for host resistance is possible and if significant gentetic differentiation is possible to identify. The aim of this project is to better understand the specific immune response to Foot-and-Mouth Disease Virus (FMDV) infection and vaccination and to identify the genetic basis of animals with high and low responder phenotypes. In FY 2012 studies were conducted on the local adaptive responses induced in cattle vaccinated and further infected through the oronasal route. Animals were intramuscularly vaccinated with serotype O1 Campus monvalent FMD oil vaccine and studied at different times post-vaccination and post-oronasal challenge. Samples were collected and analyzed using FMDV-ASC ELISpot and Interferon-gamma (IFN-y) levels through IFN-y ELISpot. Analysis of the results indicated that even though none of the vaccinated animals showed FMD clinical symptoms after viral challenge, mucosal stimulation following intramuscular administration of a high- payload FMDV-inactivated oil vaccine was poor. A modest, though consistent stimulation in all mucosal-related lymphoid tissues was detected only at 29 days post vaccination. Results indicated that after 4 weeks, vaccination may induce a basal stimulation in lymphoid tissues associated to the cattle respiratory tract. This may be related to the circulation of viral antigens or FMDV primed-immune cells to other lymph nodes in the respiratory tract. In addition, studies were conducted on the heritability factors involved in the response to FMDV vaccination. Paired whole blood samples were obtained from 377 animals in the study herd. Samples were taken the same day prior to primo vaccination in the frame of the official FMD vaccination campaigns in Argentina and at 45 days post vaccination. The animals received commercial tetravalent FMD oil vaccines formulated with inactivated FMDV from A24 Cruzeiro/Brazil/55.A/Argentina/20012, 01 Campus/Brazil/58 and C3/Indaial/Brazil/71 strains. The total FMDV- specific antibody response were determined against 3 viral strains belonging to serotypes with recent regional circulation. Anti-FMD antibody titers were analyzed to discard calves with FMDV-specific colostral antibodies from their vaccinated dams before vaccination. Analysis of the results indicated that the antibody responses induced after primo-vaccination were homogenously high for all calves assayed. In addition, the hierarchical mixed regression models showed that the sire is a random effect, and did not improve the fit of the model for any of the three virus strains. However, the sire�s breed was a significant for the thee strains, resulting in an antibody response to FMD vaccination at 45 dpv significantly lower the progeny of Jersey compared to Holstein.
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Progress 10/01/10 to 09/30/11
Outputs
Progress Report Objectives (from AD-416) The objective of this collaborative agreement is to gain a better understanding of specific immune response to Foot-and-Mouth Disease Virus (FMDV) infection and vaccination and to identify the genetic basis of animals with high and low responder phenotypes. The objectives are: 1. to describe the early steps of the specific immune response against FMDV infection, 2. to describe the immune response elicited by inactivated FMDV vaccination, and 3. to determine the heritability of the response to FMDV vaccination in cattle populations. Approach (from AD-416) 1. The early steps of specific immune response against FMDV infection will be done through infection by aerosol inoculation delivery route and subsequent virus detection of respiratory tissues through immunostaining and real-time RT-PCR will be done. Replication sites will be correlated to anti-FMDV antibody-producing cells and studied. 2. The immune response elicited by inactivated FMDV vaccination will be studied. Cells responsible for antibody production will be determined through ELISpot assay and analyzed for protective response elicted by inactivated FMDV vaccines. These cells will genotyped with the aim of correlating host genotype with host resistance/recovery after experimental infection or induction of protection post vaccination. Results obtained from in vivo experiments will then be compared to in vitro responses. 3. The heritability of response to FMDV vaccination will be conducted studies of vaccinated bovine populations in Argentina with known pedigree. Estimates of heritability will be calculated to assess host genetic influence to FMDV vaccination response. These estimates will be used to assess merit of further efforts to identify bovine strains purported to demonstrate enhanced levels of innate resistance to FMDV. This will determine if selection for host resistance is possible and if significant gentetic differentiation is possible to identify. Significant information was generated regarding the beginning of the FMDV- specific local immune responses in na�ve cattle. This includes organ and tissues involved, magnitude and isotypes of the antibodies induced and the temporal evolution of all these parameters. A manuscript describing this work is currently under preparation. In addition, a set of 334 paired samples from primo vaccinated calves were analyzed, resulting in the identification of groups of calves descending from the same sire with significantly better humoral responses to the primo vaccination. This project was monitored through email and telephone exchange and site visits to INTA.
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Progress 10/01/09 to 09/30/10
Outputs
Progress Report Objectives (from AD-416) The objective of this collaborative agreement is to gain a better understanding of specific immune response to Foot-and-Mouth Disease Virus (FMDV) infection and vaccination and to identify the genetic basis of animals with high and low responder phenotypes. The objectives are: 1. to describe the early steps of the specific immune response against FMDV infection, 2. to describe the immune response elicited by inactivated FMDV vaccination, and 3. to determine the heritability of the response to FMDV vaccination in cattle populations. Approach (from AD-416) 1. The early steps of specific immune response against FMDV infection will be done through infection by aerosol inoculation delivery route and subsequent virus detection of respiratory tissues through immunostaining and real-time RT-PCR will be done. Replication sites will be correlated to anti-FMDV antibody-producing cells and studied. 2. The immune response elicited by inactivated FMDV vaccination will be studied. Cells responsible for antibody production will be determined through ELISpot assay and analyzed for protective response elicted by inactivated FMDV vaccines. These cells will genotyped with the aim of correlating host genotype with host resistance/recovery after experimental infection or induction of protection post vaccination. Results obtained from in vivo experiments will then be compared to in vitro responses. 3. The heritability of response to FMDV vaccination will be conducted studies of vaccinated bovine populations in Argentina with known pedigree. Estimates of heritability will be calculated to assess host genetic influence to FMDV vaccination response. These estimates will be used to assess merit of further efforts to identify bovine strains purported to demonstrate enhanced levels of innate resistance to FMDV. This will determine if selection for host resistance is possible and if significant gentetic differentiation is possible to identify. In FY2010 we completed the set up of an antibody-secreting cell (ASC) ELISpot assay specific for Foot-and-Mouth Disease Virus (FMDV) to measure adaptive humoral response against FMDV induced in the respiratory tract of cattle. All these in vitro and in vivo experiments were carried out at the Institute of Virology, INTA (Argentina). The assay was initially tested in 4 steers which were challenged with virulent FMDV O1 Campos by intradermolingual (IDL) route. Animals were euthanized at 7 (na�ve) or 11 (vaccinated) days post infection (dpi) and 7 tissues were processed to be assayed by the FMDV ASC ELISpot. FMDV- specific ASCs were detected in all lymphoid tissues studied, with different reactivity patterns between na�ve and vaccinated cattle. Next, cattle infected with virulent FMDV O1 Campos by the oronasal route, following a protocol developed at ARS-PIADC, were assayed. Two animals were infected and euthanized at 3 and 6 dpi respectively, to obtain B-lymphocytes from lymphoid tissues within the bovine respiratory tract. A third uninfected steer was also assayed as negative control. Both the uninfected and the 3 dpi-infected steers did not show FMDV- specific ASC in any of the tissues studied. However, the 6 dpi-infected animal presented a very vigorous adaptive local immune response, showing a typical primary-response Ig isotype pattern. Additionally, experiments were carried out to determine the heritability of the response to FMDV vaccination in cattle populations. FMDV-specific systemic humoral responses elicited at 0 and 45 dpv in primo vaccinated calves born to vaccinated mothers were determined by lpELISA. Complete genealogic trees from these calves and HB information corresponding to bulls used for insemination in each case, were obtained for genetic characterization of the animals and sampling design. A total of 101 samples were analyzed so far, showing no statistically significant differences between steers born from different bulls. Antibodies against 2 other FMDV strains included in the vaccine (A24 Cruzeiro and A2001 Argentina) are currently assayed in this pool of samples to study whether these results are strain-related, although more samples will be obtained and analyzed during FY 2011 to get conclusive results on this study. The major accomplishment of this collaboration is the development of an ASC ELISpot assay specific for FMDV to measure adaptive humoral response against FMDV induced in the respiratory tract of cattle. The assay was tested in cattle infected with FMDV O1 Campos by the IDL or oronasal route, being able to quantify and determine the Ig isotype profile of FMDV-specific ASC elicited after infection, in all lymphoid tissues analyzed. This project was monitored through email and telephone exchange as well as site visits to INTA and ARS, PIADC.
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Progress 10/01/08 to 09/30/09
Outputs
Progress Report Objectives (from AD-416) The objective of this collaborative agreement is to gain a better understanding of specific immune response to Foot-and-Mouth Disease Virus (FMDV) infection and vaccination and to identify the genetic basis of animals with high and low responder phenotypes. The objectives are: 1. to describe the early steps of the specific immune response against FMDV infection, 2. to describe the immune response elicited by inactivated FMDV vaccination, and 3. to determine the heritability of the response to FMDV vaccination in cattle populations. Approach (from AD-416) 1. The early steps of specific immune response against FMDV infection will be done through infection by aerosol inoculation delivery route and subsequent virus detection of respiratory tissues through immunostaining and real-time RT-PCR will be done. Replication sites will be correlated to anti-FMDV antibody-producing cells and studied. 2. The immune response elicited by inactivated FMDV vaccination will be studied. Cells responsible for antibody production will be determined through ELISpot assay and analyzed for protective response elicted by inactivated FMDV vaccines. These cells will genotyped with the aim of correlating host genotype with host resistance/recovery after experimental infection or induction of protection post vaccination. Results obtained from in vivo experiments will then be compared to in vitro responses. 3. The heritability of response to FMDV vaccination will be conducted studies of vaccinated bovine populations in Argentina with known pedigree. Estimates of heritability will be calculated to assess host genetic influence to FMDV vaccination response. These estimates will be used to assess merit of further efforts to identify bovine strains purported to demonstrate enhanced levels of innate resistance to FMDV. This will determine if selection for host resistance is possible and if significant gentetic differentiation is possible to identify. Significant Activities that Support Special Target Populations INTA scientists visited ARS, PIADC and were trained in sampling of these organs and tissues in cattle and also regarding the protocols developed at PIADC for experimental infection of cattle using FMDV applied through the respiratory tract. Conditions regarding the FMDV-specific ASC ELISpot assay were set up using MAbs against FMDV and PBMC from FMDV vaccinated cattle. These experiments were performed at INTA in collaboration with the Institute of Science and Technology Cesar Milstein, depending on National Research Council of Argentina. Sanitary services responsible for 2 dairy farms in the Province of Buenos Aires were contacted in order to use animals from these farms for the above mentioned study. Dairy farms carry out a strict genetic control of their animals, thus making possible to track parents from claves born there. Also, due to production strategy of these farms, calves are born all along the year reducing restrictions related to the breeding season. Lists of calves born to mothers belonging to these farms and from bulls used to inseminate them were generated and analyzed. Also, complete genealogic trees and HB information of these bulls within the American Dairy Bulls database were obtained and made available to other participants of this project involved in genetic characterization of the animals and sampling design. Activities in this project were monitored through email and telephone exchange, as well as site visits to both ARS, PIADC and INTA.
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