Southern African Territories Research Articles

Optimization of a foot-and-mouth disease virus Southern African Territories-specific solid-phase competitive ELISA for small ruminant serum samples.

We optimized and verified a single-spot solid-phase competitive ELISA (ss-SPCE) to detect antibodies against structural proteins of Southern African Territories (SAT) serotypes of foot-and-mouth disease virus (FMDV) in small ruminants. Sera from goats vaccinated and experimentally challenged with a SAT1 FMDV pool were tested in duplicate at 4 dilutions (1:10, 1:15, 1:22.5, 1:33.8) to optimize the assay. To assess the performance of the assay in naturally infected animals, we evaluated 316 goat and sheep field sera collected during active SAT2 outbreaks. Relative to results of the virus neutralization test, the optimal serum dilution and cutoff percentage inhibition (PI) were 1:15 and 50%, respectively. At these values, the Spearman rank correlation coefficient was 0.85 (p < 0.001), and the sensitivity and specificity (95% CI) were 80.3% (72.6, 87.2) and 91.1% (84.1, 95.9), respectively. Relative to the liquid-phase blocking ELISA and the nonstructural protein ELISA, the ss-SPCE exhibited divergent performance characteristics between the goat and sheep field sera. Repeatability was better for goats, but the correlation and agreement among all 3 assays were better for the sheep sera. The prevalence of SAT2 FMDV infection in the sampled sheep was 23.6%; sampled goats were seemingly FMDV-free. The ss-SPCE is an appropriate FMDV detection tool to investigate the role of small ruminants in the epidemiology of FMD in Africa.

A TaqMan-based RT-qPCR assay for serotyping of Southern African territories (SAT) 2 strains of Foot-and-Mouth disease virus (FMDV) in Southern Africa

ObjectiveDetermining the serotype of circulating virus strains is important in implementing effective vaccination. In this study, Foot-and-Mouth Disease (FMD) Southern African territory 2 (SAT2) specific primers and TaqMan probe were designed towards rapid SAT2 detection and serotyping. The primers were tested by endpoint reverse transcription (RT) polymerase chain reaction (PCR) and quantitative PCR (RT-qPCR) using the vaccine strain SAT2035. The SAT2 serotype-specific RT-qPCR assay was compared with currently used ELISA and VP1 sequencing using Cohen’s kappa statistics.ResultsThe primers yielded amplicons of band size 190 bp during endpoint RT-PCR. When coupled with the probe, the primers reaction efficiency was determined to be 99% with an r2 value of 0.994. The results show that the SAT2 assay has comparable performance to VP1 sequencing (k = 1) and a moderate degree of agreement with ELISA (k = 0.571). The data shows that the newly designed assay could be considered for serotyping of SAT2 strains. However, for this assay to be complete there is a need to design effective SAT1 and SAT3 primers and probes that can be multiplexed to target other serotypes that co-circulate within relevant FMD endemic pools. For future implementation of the assay there is also a need to increase the number of field samples towards validation of the assay.

A review of foot-and-mouth disease status and control measures in Botswana.

Foot-and-mouth disease (FMD), an economically important disease of livestock, is endemic in Botswana. The country has been affected by this disease since the early 1930s, and FMD virus (FMDV) continues to circulate in both domestic and wild animal populations. Botswana is affected by the Southern African Territories (SAT1-3) of FMDV. Up to 80% of the income in the agricultural sector in Botswana is derived from the beef production, and about 70% of Botswana's beef exports go to the European Union (EU) market. Thus, trade restrictions caused by FMD outbreaks may result in declines in revenue. In this review, the FMD status of Botswana from 2006 to 2022 is discussed. During the report period, SAT2 was responsible for 80 out of a total of 87 FMD outbreaks, while SAT1 was responsible for 7 out of 87 outbreaks. These outbreaks were a result of SAT1 topotype I and SAT2 topotypes I, II, and III. There were no outbreaks associated with serotype SAT3 over the review span, suggesting absence of this serotype in the country, although it is still maintained in vaccines formulated for use in Botswana. Most of the outbreaks reported in this review occurred in the North West district of Botswana; an area that is heavily populated with cloven hooved wildlife. This highlights the role of wildlife-domestic animal interaction in FMD spread and maintenance. The Food and Agriculture Organization (FAO) of the United Nations has created a progressive control pathway for FMD (PCP-FMD) for the global elimination of FMD to reduce FMD-related losses. This review highlights how Botswana takes part in the PCP-FMD by putting in place control measures such as surveillance and vaccination. The review also touches on the disease control challenges such as limitations to separation of livestock with populations of buffaloes and lapses in livestock vaccination which contribute to maintenance of FMDV circulation in Botswana.

Preliminary validation of a single-spot version of a solid-phase competition ELISA for the detection of southern African territories foot-and-mouth disease serotype exposure in goats

The objective of this study was to perform a preliminary validation of a solid-phase competition ELISA (SPCE) in goats exposed to foot-and-mouth disease virus (FMDV) southern African territories (SAT) serotypes through vaccination or experimental infection. Thirty-nine goats were vaccinated with a FMDV vaccine and 37 subsequently challenged with a SAT1 virus serotype. Blood was collected every 7 days until termination at 14 days post-challenge. Single-spot SAT1 virus serotype SPCE (ss-SPCE) was performed in duplicate at two time points and a half-titration version was performed after a variable time of long-term storage. Coefficient of variation (CV) was calculated and accuracy of the ss-SPCE was estimated relative to a half-titration SPCE log10 titer of 1.6 using mixed-effect logistic regression. Additionally, sensitivity and specificity were estimated based on serological results 14-days post-challenge and at study enrolment, respectively. Three hundred and forty-two serum samples were tested in duplicate on two non-consecutive days. The median (interquartile range (IQR)) CV for the ss-SPCE for SAT1 was 2.1% (0.5, 14.3%) and 2.5% (0.6, 12.8%) for the two testing days, respectively. Median (IQR) inter-assay (different day) CV was 10.6% (2.5, 42.5%). Specificity and sensitivity of the ss-SPCE relative to the log10 titer using a 70% percentage inhibition positive threshold were 83.4% (95% confidence interval, 77.7–87.9) and 95.8% (90.7–98.2), respectively. Specificity was estimated as 100% (92.6, 100) and sensitivity as 97.3% (87.4, 99.9) when only considering serum tested at the beginning and end of the study, respectively. The SAT1 ss-SPCE is repeatable and accurate for determining FMDV serological status in goats.

Development of reverse-transcriptase, real-time PCR assays to distinguish the Southern African Territories (SAT) serotypes 1 and 3 and topotype VII of SAT2 of Foot-and-Mouth Disease Virus.

Foot-and-Mouth Disease Virus (FMDV), the causative agent of Foot-and-Mouth Disease, is a highly feared, economically devastating transboundary pathogen. This is due to the virus' extremely contagious nature and its ability to utilize multiple transmission routes. As such, rapid and accurate diagnostic testing is imperative to the control of FMD. Identification of the FMDV serotype is necessary as it provides the foundation for appropriate vaccine selection and aids in outbreak source tracing. With the vast genetic diversity, there is a desperate need to be able to characterize FMDV without relying on prior knowledge of viral serotypes. In this study, the Neptune bioinformatics tool was used to identify genetic signatures specific to each Southern African Territories (SAT) 1, 2 and 3 genomes but exclusionary to the other circulating FMDV serotypes (A, O, Asia1, and the heterologous SAT1, SAT2 and/or SAT3). Identification of these unique genomic regions allowed the design of TaqMan-based real-time reverse transcriptase PCR (rRT-PCR) primer/probe sets for SAT1, SAT2 and SAT3 viruses. These assays were optimized using prototypic FMDV cell culture isolates using the same reagents and thermocycling conditions as the FMDV pan-serotype 3D rRT-PCR assay. Cross-reactivity was evaluated in tandem with the FMDV pan-serotype 3D rRT-PCR utilizing representative strains from FMDV serotypes A, O, Asia1, SAT1, SAT2 and SAT3. The SAT1, SAT2, and SAT3 primer/probe sets were specific for the homologous serotype and exclusionary to all others. SAT1 and SAT3 primer/probe sets were able to detect several topotypes, whereas the SAT2 assay was revealed to be specific for topotype VII. The SAT2 topotype VII specificity was possibly due to the use of sequence data deposited post-2011to design the rRT-PCR primers and probes. Each assay was tested against a panel of 99 bovine tissue samples from Nigeria, where SAT2 topotype VII viruses were correctly identified and no cross-reactivity was exhibited by the SAT1 and 3 assays. These novel SAT1, SAT3 and SAT2 topotype VII rRT-PCR assays have the potential to detect and differentiate circulating FMD SAT viruses.

Viral dynamics and immune responses to foot-and-mouth disease virus in African buffalo (Syncerus caffer)

Foot-and-mouth disease (FMD) is one of the most important livestock diseases restricting international trade. While African buffalo (Syncerus caffer) act as the main wildlife reservoir, viral and immune response dynamics during FMD virus acute infection have not been described before in this species. We used experimental needle inoculation and contact infections with three Southern African Territories serotypes to assess clinical, virological and immunological dynamics for thirty days post infection. Clinical FMD in the needle inoculated buffalo was mild and characterised by pyrexia. Despite the absence of generalised vesicles, all contact animals were readily infected with their respective serotypes within the first two to nine days after being mixed with needle challenged buffalo. Irrespective of the route of infection or serotype, there were positive associations between the viral loads in blood and the induction of host innate pro-inflammatory cytokines and acute phase proteins. Viral loads in blood and tonsil swabs were tightly correlated during the acute phase of the infection, however, viraemia significantly declined after a peak at four days post-infection (dpi), which correlated with the presence of detectable neutralising antibodies. In contrast, infectious virus was isolated in the tonsil swabs until the last sampling point (30 dpi) in most animals. The pattern of virus detection in serum and tonsil swabs was similar for all three serotypes in the direct challenged and contact challenged animals. We have demonstrated for the first time that African buffalo are indeed systemically affected by FMD virus and clinical FMD in buffalo is characterized by a transient pyrexia. Despite the lack of FMD lesions, infection of African buffalo was characterised by high viral loads in blood and oropharynx, rapid and strong host innate and adaptive immune responses and high transmissibility.

Molecular Characterization of Southern African Territories 2 (SAT2) Serotype of Foot-and-Mouth Disease Virus from Nigeria in 2017 to 2018.

ABSTRACTThis report describes the nucleotide sequences of eight Southern African Territories 2 (SAT2) serotype foot-and-mouth disease virus strains from 2017 to 2018 outbreaks in cattle in Nigeria. These viruses belong to topotype VII of SAT2 and were closely related to previous isolates from Nigeria and other West African countries.

A Vaccine Strain of the A/ASIA/Sea-97 Lineage of Foot-and-Mouth Disease Virus with a Single Amino Acid Substitution in the P1 Region That Is Adapted to Suspension Culture Provides High Immunogenicity.

There are seven viral serotypes of foot-and-mouth disease virus (FMDV): A, O, C, Asia 1, and Southern African Territories 1, 2, and 3 (SAT 1–3). Unlike serotype O FMDV vaccine strains, vaccine strains of serotype A FMDV do not provide broad-range cross-reactivity in serological matching tests with field isolates. Therefore, the topotype/lineage vaccine strain circulating in many countries and a highly immunogenic strain might be advantageous to control serotype A FMDV. We developed a new vaccine strain, A/SKR/Yeoncheon/2017 (A-1), which belongs to the A/ASIA/Sea-97 lineage that frequently occurs in Asian countries. Using virus plaque purification, we selected a vaccine virus with high antigen productivity and the lowest numbers of P1 mutations among cell-adapted virus populations. The A/SKR/Yeoncheon/2017 (A-1) vaccine strain has a single amino acid mutation, VP2 E82K, in the P1 region, and it is perfectly adapted to suspension culture. The A/SKR/Yeoncheon/2017 (A-1) experimental vaccine conferred high immunogenicity in pigs. The vaccine strain was serologically matched with various field isolates in two-dimensional virus neutralization tests using bovine serum. Vaccinated mice were protected against an A/MAY/97 virus that was serologically mismatched with the vaccine strain. Thus, A/SKR/Yeoncheon/2017 (A-1) might be a promising vaccine candidate for protection against the emerging FMDV serotype A in Asia.

Symmetrical arrangement of positively charged residues around the 5-fold axes of SAT type foot-and-mouth disease virus enhances cell culture of field viruses.

Field isolates of foot-and-mouth disease viruses (FMDVs) utilize integrin-mediated cell entry but many, including Southern African Territories (SAT) viruses, are difficult to adapt to BHK-21 cells, thus hampering large-scale propagation of vaccine antigen. However, FMDVs acquire the ability to bind to cell surface heparan sulphate proteoglycans, following serial cytolytic infections in cell culture, likely by the selection of rapidly replicating FMDV variants. In this study, fourteen SAT1 and SAT2 viruses, serially passaged in BHK-21 cells, were virulent in CHO-K1 cells and displayed enhanced affinity for heparan, as opposed to their low-passage counterparts. Comparative sequence analysis revealed the fixation of positively charged residues clustered close to the icosahedral 5-fold axes of the virus, at amino acid positions 83-85 in the βD-βE loop and 110-112 in the βF-βG loop of VP1 upon adaptation to cultured cells. Molecular docking simulations confirmed enhanced binding of heparan sulphate to a model of the adapted SAT1 virus, with the region around VP1 arginine 112 contributing the most to binding. Using this information, eight chimeric field strain mutant viruses were constructed with additional positive charges in repeated clusters on the virion surface. Five of these bound heparan sulphate with expanded cell tropism, which should facilitate large-scale propagation. However, only positively charged residues at position 110-112 of VP1 enhanced infectivity of BHK-21 cells. The symmetrical arrangement of even a single amino acid residue in the FMD virion is a powerful strategy enabling the virus to generate novel receptor binding and alternative host-cell interactions.

Genetic Basis of Antigenic Variation of SAT3 Foot-And-Mouth Disease Viruses in Southern Africa.

Foot-and-mouth disease (FMD) continues to be a major burden for livestock owners in endemic countries and a continuous threat to FMD-free countries. The epidemiology and control of FMD in Africa is complicated by the presence of five clinically indistinguishable serotypes. Of these the Southern African Territories (SAT) type 3 has received limited attention, likely due to its restricted distribution and it being less frequently detected. We investigated the intratypic genetic variation of the complete P1 capsid-coding region of 22 SAT3 viruses and confirmed the geographical distribution of five of the six SAT3 topotypes. The antigenic cross-reactivity of 12 SAT3 viruses against reference antisera was assessed by performing virus neutralization assays and calculating the r1-values, which is a ratio of the heterologous neutralizing titer to the homologous neutralizing titer. Interestingly, cross-reactivity between the SAT3 reference antisera and many SAT3 viruses was notably high (r1-values >0.3). Moreover, some of the SAT3 viruses reacted more strongly to the reference sera compared to the homologous virus (r1-values >1). An increase in the avidity of the reference antisera to the heterologous viruses could explain some of the higher neutralization titers observed. Subsequently, we used the antigenic variability data and corresponding genetic and structural data to predict naturally occurring amino acid positions that correlate with antigenic changes. We identified four unique residues within the VP1, VP2, and VP3 proteins, associated with a change in cross-reactivity, with two sites that change simultaneously. The analysis of antigenic variation in the context of sequence differences is critical for both surveillance-informed selection of effective vaccines and the rational design of vaccine antigens tailored for specific geographic localities, using reverse genetics.

Complex Circulation of Foot-and-Mouth Disease Virus in Cattle in Nigeria.

Nigeria is a large densely populated country in West Africa. Most of its livestock is raised in a pastoralist production system with typical long distance migration in search of water and feed. As the demand for animal products largely exceeds the domestic production, large numbers of livestock are imported from neighboring countries without sanitary restrictions. In Nigeria, foot-and-mouth disease virus (FMDV) serotypes O, A, and Southern African Territories (SAT)2 are endemic for a long time. Clinical outbreaks of FMD due to serotype SAT1 are described again since 2015, after an absence of more than 30 years. Historically, outbreaks of FMD due to serotypes O, A, SAT1, and SAT2 were each time associated with trade of cattle entering Nigeria from neighboring countries. In the present study, tissue samples from 27 outbreaks of FMD were collected in Nigerian cattle from 2012 until 2017 in six different States and in the Federal Capital Territory. FMDV was isolated and serotyped and further characterized by VP1 sequencing and phylogenetic analysis to gain more knowledge on FMDV circulation in Nigeria. Half of the outbreaks were characterized as FMDV topotype O/EA-3, while outbreaks with other serotypes and topotypes were—in descending order—less prevalent: A/Africa/G-IV, SAT1/X, SAT2/VII, and O/WA. The high dynamics and omnipresence of FMD in Nigeria were illustrated in Plateau State where FMDV serotypes O, SAT1, and SAT2 were isolated during the course of the study, while at some point in the study, outbreaks due to FMDV serotype A were observed in three remote States. The genetic and phylogenetic analysis suggests a mixed origin of FMD outbreaks. Some outbreaks seem to be caused by sustained local transmission of FMDV strains present in Nigeria since a number of years, while other outbreaks seem to be related to recent incursions with new FMDV strains. The role of African buffaloes in the etiology of FMD in Nigeria is unclear, and sampling of wildlife is needed. The results of the present study suggest that systematic sample collection is essential to understand the complex concomitance of FMDV strains in Nigeria and essential to support the implementation of a vaccination-based control plan.

Vaccine strain of O/ME-SA/Ind-2001e of foot-and-mouth disease virus provides high immunogenicity and broad antigenic coverage

Foot-and-mouth disease (FMD) is an economically devastating animal disease. There are seven serotypes, A, O, C, Asia 1, Southern African Territories 1, 2, and 3 (SAT1, SAT2, and SAT3), among which serotype O shows the greatest distribution worldwide. Specifically, the O/ME-SA/Ind-2001 lineage, which was reported in India in 2001, has since emerged worldwide, with the O/ME-SA/Ind-2001d and O/ME-SA/Ind-2001e sublineages recently emerging in North Africa, Middle East Asia, Southeast Asia, and East Asia. The antigenic relationship (r1) value for the O1 Manisa and O/Mya-98 lineage inactivated vaccine against various O/ME-SA/Ind-2001 lineages of FMDV isolates, were matching (r1 > 0.3) or non-matching (r1 < 0.3), indicating that the vaccine based on the O/ME-SA/Ind-2001 lineage FMDV, is valuable. In this study, we developed a new vaccine strain, O/SKR/Boeun/2017 isolate, belonging to the O/ME-SA/Ind-2001e sublineage as an outbreak of this sublineage occurred in 2017 in the Boeun county of the Republic of Korea (O/SKR/Boeun/2017). This experimental vaccine exhibited high immunogenicity in pigs and cattle and was antigenically matched with representative FMDV lineages (ME-SA, O/ME-SA/PanAsia, O/SEA/Mya-98, and O/Cathay) in Asia, as demonstrated by two-dimensional virus neutralization tests (2D-VNT). In addition, a 100% survival rate in C56BL/6 mice vaccinated with 1/15 of a pig dose was observed following challenge with FMDV O/VIT/2013 (O/ME-SA/PanAsia) at 10 days post-vaccination. Further, we analyzed the major antigenic sites of the O/SKR/Boeun/2017 vaccine strain as well as other viruses, by 2D-VNT. These results suggest that the O/ME-SA/Ind-2001e sublineage is a promising vaccine strain candidate in Asia, and other countries, for protection against the emerging FMDV.

Diagnostic and Epitope Mapping Potential of Single-Chain Antibody Fragments Against Foot-and-Mouth Disease Virus Serotypes A, SAT1, and SAT3.

Foot-and-mouth disease (FMD) affects cloven-hoofed domestic and wildlife animals and an outbreak can cause severe losses in milk production, reduction in meat production and death amongst young animals. Several parts of Asia, most of Africa, and the Middle East remain endemic, thus emphasis on improved FMD vaccines, diagnostic assays, and control measures are key research areas. FMD virus (FMDV) populations are quasispecies, which pose serious implications in vaccine design and efficacy where an effective vaccine should include multiple independent neutralizing epitopes to elicit an adequate immune response. Further investigation of the residues that comprise the antigenic determinants of the virus will allow the identification of mutations in outbreak strains that potentially lessen the efficacy of a vaccine. Additionally, of utmost importance in endemic regions, is the accurate diagnosis of FMDV infection for the control and eradication of the disease. To this end, a phage display library was explored to identify FMDV epitopes for recombinant vaccines and for the generation of reagents for improved diagnostic FMD enzyme-linked immunosorbent assays (ELISAs). A naïve semi-synthetic chicken single chain variable fragment (scFv) phage display library i.e., the Nkuku® library was used for bio-panning against FMD Southern-African Territories (SAT) 1, SAT3, and serotype A viruses. Biopanning yielded one unique scFv against SAT1, two for SAT3, and nine for A22. SAT1 and SAT3 specific scFvs were exploited as capturing and detecting reagents to develop an improved diagnostic ELISA for FMDV. The SAT1 soluble scFv showed potential as a detecting reagent in the liquid phase blocking ELISA (LPBE) as it reacted specifically with a panel of SAT1 viruses, albeit with different ELISA absorbance signals. The SAT1svFv1 had little or no change on its paratope when coated on polystyrene plates whilst the SAT3scFv's paratope may have changed. SAT1 and SAT3 soluble scFvs did not neutralize the SAT1 and SAT3 viruses; however, three of the nine A22 binders i.e., A22scFv1, A22scFv2, and A22scFv8 were able to neutralize A22 virus. Following the generation of virus escape mutants through successive virus passage under scFv pressure, FMDV epitopes were postulated i.e., RGD+3 and +4 positions respectively, proving the epitope mapping potential of scFvs.

Assessment of the sensitivity of primary cells and cell lines to the Southern African Territories (SAT) serotypes in the diagnosis of foot-and-mouth disease virus

Virus isolation is the gold standard for foot-and-mouth disease virus (FMDV) detection in diagnostic procedures. This technique is heavily reliant on the use of sensitive cells for rapid and accurate detection of FMDV. To investigate the sensitivity of RM (primary lamb kidney cells), BHK-21 (baby hamster kidney cells) and IR-P1 (a derivative of female pig kidney cells) to infection with FMDV of the Southern African Territories (SAT) serotypes, we examined the virus concentration required to induce cytopathic effect (CPE) on each cell type. The results suggested that sensitivity of RM and IR-P1 cells was high and not significantly different (P < 0.05). BHK-21 however, exhibited low sensitivity to the strains used. Comparisons of three batches of each cell type were also done to establish the consistency of the sensitivity of these cells to FMDV infection. IR-P1 and BHK-21 cell batches gave consistent results for all samples used whereas RM cells showed significant differences (P > 0.05) between batches. TCID 50/ml was used to determine the viral titre required to induce CPE. IR-P1 cell line proved to have consistently higher TCID50/mL for all cell batches while RM cell batches displayed a difference in TCID50/mL values. The IR-P1 cell line was concluded to be a good cell culture system for virus isolation as it showed relatively high and reproducible sensitivity to all the FMDV strains used. The findings of this study indicate that the use of IR-P1 cell line could be considered for FMDV diagnostic work.

Efficacy of a foot-and-mouth disease vaccine against a heterologous SAT1 virus challenge in goats

Goats are susceptible to infection with foot-and-mouth disease virus (FMDV), but their role in the epidemiology of the disease and response to vaccination is poorly understood. In southern Africa, FMDV serotypes Southern African Territories (SAT) 1, 2 and 3 are known to be endemic. In this study, we evaluated the efficacy of a pentavalent FMD vaccine in goats against heterologous challenge with a pool of field SAT1 FMDV. Forty FMD sero-negative goats (6–12 months of age) of mixed sexes were randomly allocated to one of five treatment groups: full cattle dose (2 ml), 1/3rd (0.67 ml), 1/6th (0.33 ml), 1/12th (0.16 ml) or unvaccinated placebo control. Goats were vaccinated with an inactivated pentavalent FMD vaccine containing serotypes SAT1, SAT2 and SAT3 on day 0 and revaccinated at day 20 post vaccination. Thereafter, thirty-four goats were challenged by tongue inoculation at day 41 post-vaccination using 104.57 50% tissue culture infective dose (TCID50) FMDV SAT1 pool. Animals were examined daily and clinical signs were scored. Rectal temperatures were measured daily, with temperatures ≥40 °C defined as fever. Clinical specimens (nasal, oral and rectal swabs) were collected on days 0, 2, 4 and 6 post challenge. Viral shedding was determined using reverse-transcriptase real-time PCR. None of the goats vaccinated with the full cattle dose developed secondary lesions. All vaccinated groups had lower temperatures compared to the unvaccinated controls (P < 0.001). Based on RT-PCR results, goats in the unvaccinated control group shed more virus compared to all groups except for 1/12th (P < 0.05), while goats in the full dose group shed less virus than goats in the 1/12th and the unvaccinated control group (P < 0.05). The results suggest that the 1/3rd (0.67 ml) dose of the vaccine is sufficient to reduce viral shedding after heterologous challenge with a FMDV SAT1 pool.

Pathogenesis, biophysical stability and phenotypic variance of SAT2 foot-and-mouth disease virus

Foot-and-mouth disease (FMD) is a highly contagious vesicular disease of cloven-hoofed animals, which severely decreases livestock productivity. FMD virus (FMDV), the causative agent, initiates infection by interaction with integrin cellular receptors on pharyngeal epithelium cells, causing clinical signs one to four days after transmission to a susceptible host. However, some Southern African Territories (SAT) viruses have been reported to cause mild or subclinical infections that may go undiagnosed in field conditions and are likely to be more common than previously expected. The studies presented here demonstrate that not all SAT2 viruses are equally virulent in cattle. The two SAT2 viruses, ZIM/5/83 and ZIM/7/83, were both highly attenuated in cattle, as evidenced by the mild clinical signs observed after needle challenge, while two incongruent SAT2 viruses showed significantly different clinical signs in challenged cattle. We then explored the ability of the SAT2 viruses to infect different cell types with defined receptors that are utilised by FMDV and found differences in their ability to lyse cells in culture and to compete in a controlled cell culture environment. The population sequence variation between ZIM/5/83 and ZIM/7/83 revealed multiple sites of single nucleotide variants of low frequency between the predominant virus populations, as could be expected from the genome of an RNA virus. An assessment of the biophysical stability of SAT2 virions during acidification indicated that the SAT2 virus EGY/09/12 was more resilient to acidification than the ZIM/5/83 and ZIM/7/83 viruses; however, whether this difference relates to differences in virulence in vivo is unclear. This study is a consolidated view of the key findings of SAT2 viruses studied over a 14-year period involving many different experiments.

Characterization of SAT2 foot-and-mouth disease 2013/2014 outbreak viruses at the wildlife-livestock interface in South Africa.

The Southern African Territories (SAT)-type foot-and-mouth disease viruses (FMDV) are endemic to the greater Kruger National Park (KNP) area in South Africa, where they are maintained through persistent infections in African buffalo. The occurrence of FMDV within the Greater KNP area constitutes a continual threat to the livestock industry. To expand on knowledge of FMDV diversity, the genetic and antigenic relatedness of SAT2-type viruses isolated from cattle during a FMD outbreak in Mpumalanga Province in 2013 and 2014 were investigated. Cattle from twelve diptanks tested positive on polymerase chain reaction (PCR), and molecular epidemiological relationships of the viruses were determined by VP1 sequencing. Phylogenetic analysis of the SAT2 viruses from the FMD outbreak in Mpumalanga in 2013/2014 revealed their genetic relatedness to other SAT2 isolates from topotype I (South Africa, Zimbabwe and Mozambique), albeit genetically distinct from previous South African outbreak viruses (2011 and 2012) from the same topotype. The fifteen SAT2 field isolates clustered into a novel genotype with ≥98.7% nucleotide identity. High neutralization antibody titres were observed for four 2013/2014 outbreak viruses tested against the SAT2 reference antisera representative of viruses isolated from cattle and buffalo from South Africa (topotype I) and Zimbabwe (topotype II). Comparison of the antigenic relationship (r1 values) of the outbreak viruses with reference antisera indicated a good vaccine match with 90% of r1 values>0.3. The r1 values for the 2013/2014 outbreak viruses were 0.4 and above for the three South African vaccine/reference strains. These results confirm the presence of genetic and antigenic variability in SAT2 viruses and suggest the emergence of new variants at the wildlife-livestock interface in South Africa. Continuous characterization of field viruses should be performed to identify new virus strains as epidemiological surveillance to improve vaccination efforts.

Impact of foot-and-mouth-disease on goat behaviour after experimental infection with serotype SAT1 virus

Infectious diseases and parasitic infestations can cause a set of non-specific clinical signs, such as increased body temperature and resting, and a decrease in food intake. These physiological and behavioural changes have an adaptive function facilitating defences against the pathogen and to support immune functions. These so-called’ sickness behaviours’ can also be used as an early detection tool for disease. Foot-and-mouth disease (FMD) still causes great economic losses in endemic countries, especially to smallholder farmers. The aim of this study was to determine if behavioural changes in goats can be used as an early indicator of FMD virus (FMDV) infection. The efficacy of a Southern African Territories (SAT) FMD vaccine was studied on forty South African indigenous goats. Changes in daily activities (resting, feeding, walking), as well as social behaviours (social resting, social feeding, dominance behaviours) were recorded and then compared over time and between clinically affected and unaffected goats. Pedometers were used to estimate average daily steps and to compare between groups of study animals.Eleven goats developed clinical signs of FMD, as well as non-FMD related sicknesses during the course of the study. Overall walking and resting behaviours were not significantly affected by the presence of FMD related clinical signs (p > 0.05). However, during the time of FMDV infection, social resting increased significantly (p < 0.001). Although goats developed FMD lesions on lips and tongues, percentage of time feeding was not affected (p = 0.762), suggesting that the study goats did not perceive the oral lesions as an important disturbance. Similarly, the number of steps did not consistently decrease in the presence of FMD-associated foot lesions. When affected by non-FMD related sicknesses, animals did not have an overall reduction in the time spent feeding (p = 0.867). However, goats affected with non-FMD conditions reduced the amount of social feeding (p = 0.002), potentially avoiding energetically costly competition at the feeding points. Overall, goats affected with FMD did not show more sickness behaviour, suggesting that FMDV infection in goats might not lead to obvious and therefore, easily detectable behavioural changes. This might have implications for farmers and animal health personnel, as individual goats infected with FMDV might be undetected within a flock due to the absence of obvious sickness behaviours, and the virus can therefore be spread more easily between herds through animal movements.

Mutations in foot and mouth disease virus types A and O isolated from vaccinated animals.

Foot and mouth disease (FMD) is responsible for serious economic losses in Egypt. Although vaccination is practised as the main control strategy, failure of vaccination has been reported in many cases, which can be due to a number of factors. Selection of FMD antigenic variants under the immune pressure of partially immunised hosts has been previously recorded. This study was designed to isolate and characterise foot and mouth disease viruses (FMDVs) circulating in Egyptian vaccinated animals. Serotype O, A and Southern African Territories (SAT) 2 FMDVs were detected in different Egyptian governorates during 2015, 2016 and 2017. The successful isolation of 15 FMDVs of the three serotypes is reported in this paper. Phylogenetic analysis based on the viral protein (VP) 1 gene showed that all serotype O isolates had East Africa (EA)-3 topotypes. There was variation in 15-17 amino acids between the serotype O isolates of 2015 and those of 2016 and 2017. The serotype A isolates belonged to the A-Iran-05 lineage, with the exception of one isolate of 2016 which clustered with the African strains of G-IV. Serotype SAT2 FMDV was detected in two samples of 2017 and both were of lineage Alx-12 of topotype VII. The virus neutralisation test using sera raised against vaccine strains confirmed the serotyping of the isolates and determined the antigenic relatedness between the isolates and the currently used vaccine strains. A decrease in the neutralising antibody titre of some serotype O and A isolates could be attributed to mutation in critical amino acids in the neutralising antigenic sites. Hence, this work supports previous studies describing the significance of amino acid substitutions within the antigenic sites of the virus in antibody neutralisation and immune escape.

Production of foot-and-mouth disease virus SAT2 VP1 protein

The seven serotypes of foot-and-mouth disease virus (FMDV) differ on the surface exposed regions on the VP1, 2 and 3 proteins. Amongst the three, the VP1 protein has been produced the most for use in serotyping assays for some of the Euro-Asian serotypes. In this study the VP1 protein of the FMDV SAT2/ZIM/7/83 was expressed in Escherichia coli BL21 cells in Luria broth and EnPresso® B media in shake flasks. Production was further developed and the VP1 protein was produced at 2.15 g L−1 in fed-batch fermentations at 2 L scale. The protein formed insoluble inclusion bodies that were isolated, denatured and refolded. When tested in ELISA, the protein was found to be highly reactive with serum from a SAT2 vaccinated guinea pig, and not reactive to SAT1 and SAT3 antisera. These results open avenues to evaluate recombinantly expressed VP1 proteins for differentiation of the three Southern African Territories serotypes of FMDV that co-occur in Southern and East Africa. In addition, this could mitigate the need for employing virus as reagent, or having to raise reagent antibodies.