Thesis (MMedVet (Gyn))--University of Pretoria, 2005. / Includes bibliographical references.

The Foot-and-mouth disease virus (FMDV) affects cloven-hoofed animals and is endemic in most parts of Africa, South America and southern Asia. South Africa is considered a FMDV-free zone but the virus is maintained within the wildlife in the Kruger National Park (KNP), making mitigation of outbreaks a high priority. Diagnostic methods are usually costly due to the high production cost of the reagents used, meaning that regular monitoring and diagnosis of animals around the KNP for FMDV is expensive due to the large amounts of serum continuously being tested. I propose an alternative plant expression platform for the local production of more cost effective diagnostic reagents capable of distinguishing between infected and vaccinated animals (DIVA). I selected the non-structural 3ABC polyprotein of FMDV to express, as it is a suitable candidate as a coating antigen in a competitive enzyme linked immunosorbent assay (C-ELISA) for the detection of neutralizing antibodies in livestock sera. I also chose other variations of the full polyprotein (3AB, 3AB1 and 3B) for expression as they have previously been shown to be effective in FMDV diagnosis. I also selected a second reagent to be expressed: this was the CRAb-FM27 single chain variable fragment (scFv), which binds a 3B epitope on the 3ABC polyprotein and has previously shown to be effective as a competing antibody in a C-ELISA. The 3B antigen and the scFv were successfully expressed and purified from N. benthamiana, which to my knowledge is the first time either has been shown. The plant produced scFv successfully bound the 3B antigen in an I-ELISA. Separately, the plant produced 3B antigen could be used to successfully differentiate FMDV infected and vaccinated guinea pig serum in an I-ELISA. However, testing of these reagents in tandem within a C-ELISA to DIVA sera was inconclusive, and further research is required to optimise C-ELISA conditions.

Please read the abstract in the section 00front of this document / Dissertation (MSc (Microbiology))--University of Pretoria, 2005. / Microbiology and Plant Pathology / unrestricted

Foot-and-mouth disease (FMD) is a highly contagious disease caused by the FMD virus (FMDV) belonging to the Picornaviridae family. The virus affects cloven-hoofed animals and occurs as seven immunologically distinct serotypes where six of the seven serotypes occur in Africa. This fact, as well as the role of wildlife in virus maintenance, makes eradication and control of FMDV in Africa difficult. Thus, it is imperative to attain more information regarding the genetic diversity of FMD viruses prevalent on the African continent to further our knowledge of the virus as well as to enable better control strategies and the development of improved vaccines. Sufficient genetic information regarding the Leader (L) and complete capsid-coding, P1, region of serotype A and O viruses prevalent on the African continent is lacking, although the SAT isolates have been extensively characterised in the past. In this study the sequence of the L/P1-coding region was successfully determined using a genome-walking approach for a small number of A and O viruses recovered from outbreaks isolated from various species in East and West Africa over the last 33 years. Phylogenetic analysis of the P1 and capsid-coding regions 1A, 1B, 1C and 1D revealed that the African isolates grouped strictly according to serotype and geographic region which indicated the possibility of transboundary spread of the virus within East and West African countries respectively. In contrast, phylogenetic analysis of the non-structural, Lpro-coding region revealed a different tree topology compared to the capsid-coding regions for the A and O isolates with sub-grouping according to serotype and geographic regions was less apparent. The relatedness between the serotype A and O L region might be the result of genetic recombination. The inter and intratypic nucleotide and amino acid variation of the A and O isolates revealed that the most variable capsid-coding region was the externally located VP1 whilst the internally located VP4 capsid protein was the most conserved. The observed variation is in agreement with other studies and reflects the selective pressures on these proteins which either allow or prevent the occurrence of genetic changes for structural constraints or immune escape. Surprisingly, the L protease-encoding region also displayed a high degree of variation. A detailed analysis of the L/P1 amino acid alignment of the A and O isolates revealed that although the extent of variation is high in these regions, the amino acids identified in previous studies as important for FMDV structure (for the capsid-coding regions) and function were found to be conserved, indicating that the virus has adapted itself to elude the host immune response without affecting its vital functional and structural abilities. Additionally, it was observed that the amino acid residues identified as being important for FMDV attaching to the host cell receptors e.g. the RGD amino acid motif of VP1 was highly conserved for all isolates. To further investigate the FMDV-receptor interaction, RT-PCRs were developed to examine the mRNA expression level of the known FMDV receptors. The â integrins that facilitate FMDV cell entry i.e. â1, â3, â6, â8 and heparan sulphate proteoglycans (HSPG) were investigated in susceptible cell lines used for FMDV vaccine production i.e. IB-RS-2 and BHK-21. The RT-PCRs were successfully developed and optimised. The results showed that the mRNA expression levels were variable for all receptor cDNAs tested across 36 passage levels of IB-RS-2 and BHK-21 cells. No distinct differences in virus susceptibility for three FMDV strains with continuous cell passage of IB-RS-2 and BHK-21 cells at passage levels 5, 21 and 36 could be found. The information gained from this study regarding the viral L and P1 region genetic diversity, and phylogenetic analysis has indeed impacted on our understanding of FMDV African viruses. Additionally, the investigation of the FMDV receptor mRNA expression levels and virus susceptibility on two cell lines with continuous cell passage has proved a vital starting point to determine the possible receptors expressed on the surface of cells used by the vaccine production division at the ARC-OVI-TADP and forms the basis for further investigations of the FMDV receptors on the protein level and the development of a real-time RT-PCR for FMDV receptor expression. / Dissertation (Msc)--University of Pretoria, 2008. / Veterinary Tropical Diseases / unrestricted

No description available.

Foot-and-mouth disease virus (FMDV) is one of the most contagious viruses of animals and is recognised as the most important constraint to international trade in animals and animal products. Two fundamental problems remain to be understood before more effective control measures can be put in place. These problems are the FMDV „carrier state‟ and the short duration of immunity after vaccination which contrasts with prolonged immunity after natural infection. The aim of this thesis was to study the interaction between FDMV and cells in lymphoid tissue in the natural bovine host, in order to improve our understanding of the protective immune response. Using laser capture microdissection in combination with quantitative real-time reverse transcription polymerase chain reaction, immunohistochemical analysis and corroborated by in situ hybridization, it is shown that FMDV locates rapidly to, and is maintained in, the light zone of germinal centres following primary infection of naïve cattle. Maintenance of non-replicating FMDV in these sites may represent a source of persisting infectious virus and also contribute to the generation of long-lasting antibody responses against neutralising epitopes of the virus. The role of T-lymphocyte subsets in recovery from FMDV infection in calves was investigated by administering subset-specific mouse monoclonal antibodies. Depletion of circulating CD4+ or WC1+ γδ T cells was achieved for a period extending from before challenge to after resolution of viraemia and peak clinical signs, whereas CD8+ cell depletion was only partial. Depletion of CD4+ cells was also confirmed by analysis of lymph node biopsies 5 days post-challenge. Depletion with anti-WC1 and anti-CD8 antibodies had no effect on the kinetics of infection, clinical signs and immune responses following FMDV infection. Three of the four CD4+ T-cell-depleted calves failed to generate an antibody response to the non-structural polyprotein 3ABC, but generated a neutralising antibody response similar to that in the controls, including rapid isotype switching to IgG antibody. These data suggest that antibody responses to sites on the surface of the virus capsid are T cell-independent whereas those directed against the non-structural proteins are T cell-dependent. CD4 depletion was found to substantially inhibit antibody responses to the G-H peptide loop VP1135-156 on the viral capsid, indicating that responses to this particular site, which has a more mobile structure than other neutralising sites on the virus capsid, are T cell-dependent. Depletion of CD4+ T cells had no adverse effect on the magnitude or duration of clinical signs or clearance of virus from the circulation. In conclusion, CD4+ T-cell-independent antibody responses play a major role in the resolution of primary infection with FMDV in cattle.

The goal of the work presented herein was to further our understanding of Bovine Leukocyte Antigen (BoLA) class I diversity of Holstein cattle and develop tools to measure class I restricted T cell responses to intracellular pathogens such as foot and mouth disease virus (FMDV) following vaccination. BoLA is a highly polymorphic gene region that allows the bovine immune system to differentiate pathogen-infected cells from healthy cells. Immune surveillance by CD8+ T cells plays an important role in clearing viral infections. These CD8+ T cells recognize BoLA class I molecules bearing epitopes (antigenic peptides) of intracellular origin in their peptide binding groove. Polymorphisms in the peptide binding region of class I molecules determine affinity of peptide binding and stability during antigen presentation. Different antigen peptide motifs are associated with specific genetic sequences of class I molecules. In order to better understand the adaptive immune response mediated by BoLA molecules, technologies from human medicine such as high-throughput sequencing, biochemical affinity and stability assays, tetramers and IFN-γ ELIspot assays could be applied. Therefore, it was hypothesized that we can translate these technologies from the study of human T cell responses to the study of cattle immunity. The first objective was to establish a comprehensive method for genotyping BoLA of Holstein cattle by using Illumina MiSeq, Sanger sequencing and polymerase chain reaction sequence-specific primers (PCR-SSP) (See Chapter 2). This is an important first step in order to study the BoLA restricted immune responses following FMDV vaccination. The second objective was to define the FMDV capsid protein peptide repertoire bound by BoLA class I molecules using bioinformatics and biochemical affinity and stability assays to facilitate the identification of T cell epitopes (See Chapter 3). The third objective was to demonstrate clonal T cell expansion for specific epitope polypeptides using ex-vivo multi-color flow cytometric MHC-epitope complexes (tetramers), followed by IFN-γ production measured by an ELIspot assay to quantify and define the antigen specific response of Holstein cattle to FMDV vaccination (see Chapter 4). In this, my dissertation studies aimed to improve our understanding of the BoLA class I restricted T-cell responses to candidate FMDV vaccines in Holstein cattle. In this manner, my research will improve animal health through the production of assays for characterizing the bovine immune response to intracellular pathogens and enhance vaccine design leading to improved biologicals to protect cattle from devastating infectious diseases.

Master of Science / Department of Agricultural Economics / Ted C. Schroeder / The livestock industry is susceptible to several diseases, of which Foot and Mouth Disease (FMD) is one. FMD is neither a fatal nor zoonotic animal disease, but most animals less than one year of age are killed in about 80% of cases. FMD also causes reductions in yield and milk production. FMD is recognized as an economic disease because any outbreak will lead to a drastic reduction in the export market. This study is centered on livestock production in mid-western United States. The study incorporated the result from an epidemiology model into an equilibrium displacement model; this is used to determine the economic impact of the FMD outbreak on both consumers and producers. Three vaccination-to-die scenarios were simulated. Each scenario had 200 disease spread simulation runs. The economic impact results were presented with normal distribution curves in order to see how the economic impacts were distributed across the 200 runs in each scenario. Scenario 14 with 50 and 80 herds vaccination capacity at 22 and 40 days respectively, coupled with 50 km vaccination zone has the lowest negative impact on both consumer and producers. The diseases lasted for shorter period of time in scenario 14 than scenarios 2 and 12. Scenario 14 also has least number of animals killed. It can be concluded from the equilibrium displacement outcomes that the best mitigation strategy for the control of FMD is to have a large vaccination zone area, and increment in the vaccination capacity will also curb the disease on time.

Foot-and-mouth disease (FMD) is one of the most contagious infectious diseases of domestic and wild cloven-hoofed animals, particular in cattle, sheep, pigs, goats and domestic buffalo, as well as wild ruminants such as deer. In Taiwan, there was a severe outbreak of FMD after more than 60 years freedom from the disease. The virus strain, O Taiwan 97 from the March 1997 outbreak of FMD in Taiwan, however, has been shown to have a species-specific adaptation to pigs. Although there are 7 distinct serotypes of FMD found in different regions of the world, this study focuses on the pig-adapted type O strain of FMD. After the FMD outbreak commenced in Taiwan, the spread of disease was very rapid and the whole of the western parts of Taiwan was affected within a few days after the diagnosis of FMD was confirmed. In some situations airborne transmission of FMD virus was suspected and it was speculated that this was the explanation for such rapid spread in Taiwan. Therefore, studies were conducted to investigate the transmissibility of O Taiwan/97 FMDV to susceptible pigs by direct and indirect spread including airborne spread in an enclosed animal house. This study showed that pigs in direct contact with challenged pigs became infected but none of the close-contact pigs became infected. These experiments clearly demonstrated that the pig adapted strain O Taiwan/97 was only efficiently transmitted by direct contact. This indicates that effective control against future outbreaks of pig adapted FMDV strains could be achieved by restriction of pig movement and stamping out if the outbreak has been detected in the early stages and prior to the movements of pigs from the infected premises. The measures used to control the Taiwanese FMD outbreak in 1997 were initially the slaughter of whole herds in the infected premises. However, with the rapid spread and large numbers of cases, the decision was taken to use universal compulsory vaccination of pig herds to control the outbreak when sufficient supply of vaccines was organized. Type O FMD vaccines were imported from a number of major FMD vaccine manufactures from around the world. Initially, vaccine efficacy for the imported vaccines was tested by measurement of neutralizing antibody titers in vaccinated pigs. To establish the relationship between serum neutralizing titers and protection from foot and mouth disease in pigs after vaccination, challenge studies were conducted with O/Taiwan/97 FMD in vaccinated pigs. Additionally, antibody responses to structural (neutralizing antibody) and non-structural proteins (NSP) were evaluated in vaccinated pig herds after primary and secondary vaccination in herds infected before and after vaccination. In order to be able to monitor the circulation of virus in vaccinated pig populations, valid diagnostic kits based on the detection of antibody against NSP were required. These tests needed to be evaluated against pig sera derived from challenge studies and natural FMD outbreaks. Three commercially available ELISAs (Cedi, UBI and Checkit), which were available to differentiate infected from vaccinated pigs, were tested and results showed that the Cedi test had the optimal sensitivity and specificity for pig adapted type O FMD testing. This test was used to retrospectively evaluate the sera collected from infected and non-infected pig herds collected sequentially in the year after the 1997 FMD outbreak in Taiwan. These studies also showed that the early vaccines used, stimulated NSP antibody production in swine herds that were vaccinated but not infected. This resulted in the requirement for purified FMD vaccines to be used when monitoring programs for FMD infection by NSP testing were in place. In these studies, it was also demonstrated that the purified FMD vaccines used later in the control program did not induce NSP antibody after multiple double dosage to pigs. Although clinical FMD appeared to be successfully controlled with vaccination program in Taiwan it was essential for the eradication plan to maintain active surveillance for NSP reactors in the pig population. The UBI and Cedi NSP kits were applied as screening and confirmatory tests, respectively, to pig sera collected in auction markets distributed around Taiwan to monitor for evidence of the circulation of FMD virus. Herds with positive reactors were followed-up by clinical inspection and 15 sera from suspected herds were further sampled. Negative results were obtained from all these investigation. With the absence of clinical outbreaks and the lack of evidence of FMDV circulation in the field from the NSP reactor surveillance, the Taiwanese government has progressed the eradication plan to a progressive cessation of vaccination, commencing with banning of vaccination on one isolated island in December 2006. The absence of outbreaks on that island, paved the way for further cessation of FMD vaccination in Taiwan from July 2008.

An outbreak of Foot and Mouth disease in Canada would result in the closing of borders to trade in meat and livestock between Canada and the US. The loss of export market access would result in losses to Canadian producers and negatively affect Canada’s reputation as a trading partner. Under a Regionalization Policy, trade could be allowed from disease-free regions of Canada during an outbreak. This would allow a limited amount of trade to continue and mitigate the losses to producers in uninfected areas. This thesis examined scenarios that involve various degrees of regionalization to determine the effects on producers, consumers and taxpayers. A partial- equilibrium model is used to determine the impact on economic welfare under each scenario and comparisons are made to help evaluate the relative outcomes of policies towards regionalization.