1a.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.
1b.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 (naïve) 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 naïve 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.