A Five-Year Retrospective Study of Foot-and-Mouth Disease Outbreaks in Southern Africa, 2014 to 2018

Foot-and-mouth disease (FMD) virus (FMDv), like other ribonucleic acid (RNA) genome viruses, has a tendency to mutate rapidly. As such, available vaccines may not confer enough cross-protection against incursion of new lineages and sublineages. This paper is a retrospective study to determine the topotypes/lineages that caused previous FMD outbreaks in 6 southern African countries and the efficacy of the current vaccines to protect cattle against them. A total of 453 bovine epithelial tissue samples from 33 FMD outbreaks that occurred in these countries from 2014 to 2018 were investigated for the presence of FMDv. The genetic diversity of the identified Southern African Type (SAT)-FMD viruses was determined by comparing sequences from outbreaks and historical prototype sequences. Of the 453 samples investigated, 176 were positive for four FMDv serotypes. Out of the 176 FMD positive cases there were 105 SAT2 samples, 32 SAT1 samples, 21 SAT3 samples, and 18 serotype O samples. Phylogenetic analysis grouped the SATs VP1 gene sequences into previously observed topotypes in southern Africa. SAT1 viruses were from topotypes I and III, SAT2 viruses belonged to topotypes I, II, III, and IV, and SAT3 viruses were of topotypes I and II. Vaccine matching studies on the field FMDv isolates produced r1-values greater than or equal to 0.3 for the three SAT serotypes. This suggests that there is no significant antigenic difference between current SAT FMD vaccine strains and the circulating SAT serotypes. Therefore, the vaccines are still fit-purpose for the control FMD in the region. The study did not identify incursion of any new lineages/topotypes of FMD into the sampled southern African countries.

Immuno-Efficacy of Multi-Epitope Chimeric Peptides Against Foot and Mouth Disease Virus: Potential Vaccine Candidates for Newly Emerged Serotype O and A

Artificially designed, chimeric peptide-based recombinant vaccines are novel approaches to combat the phylogenetically diverse Foot and Mouth Disease (FMD) Virus (FMDV) strains. Among seven distinct serotypes, only serotype O and A are dominantly circulating in Bangladesh and neighbouring countries of Asia, where transboundary transmission, recurrent outbreaks and emergence of novel lineages FMDV are highly prevalent. The objective of this study was to develop multi-epitope recombinant peptides, procuring immunogenicity against circulating diverse subtypes of FMDV serotype O and A. Two chimeric peptides, named B1 (41.0 kDa) and B3 (39.3 kDa), have been designed to incorporate potential B-cell and T-cell epitopes selected from multiple FMDV strains, including previously reported and newly emerged sub-lineages. After expression, characterization and immunization of guineapigs with considerable antigen load of B1 and B3 followed by the serological assays revealed the significant protective immunogenicity, developed from the higher (100 µg) doses of both antigens, against most of the currently prevalent serotype O and A strains of FMDV. The efficient expression, antigenic stability, and multivalent immunogenic potency of the chimeric peptides strongly indicate their credibility as novel vaccine candidates for FMDV serotypes O and A circulating in Bangladesh and surrounding territories.

Isolation and characterization of porcine monoclonal antibodies revealed two distinct serotype-independent epitopes on VP2 of foot-and-mouth disease virus

Pigs are susceptible to foot-and-mouth disease virus (FMDV), and the humoral immune response plays an essential role in protection against FMDV infection. However, little information is available about FMDV-specific mAbs derived from single B cells of pigs. This study aimed to determine the antigenic features of FMDV that are recognized by antibodies from pigs. Therefore, a panel of pig-derived mAbs against FMDV were developed using fluorescence-based single B cell antibody technology. Western blotting revealed that three of the antibodies (1C6, P2-7E and P2-8G) recognized conserved antigen epitopes on capsid protein VP2, and exhibited broad reactivity against both FMDV serotypes A and O. An alanine-substitution scanning assay and sequence conservation analysis elucidated that these porcine mAbs recognized two conserved epitopes on VP2: a linear epitope (2KKTEETTLL10) in the N terminus and a conformational epitope involving residues K63, H65, L66, F67, D68 and L81 on two β-sheets (B-sheet and C-sheet) that depended on the integrity of VP2. Random parings of heavy and light chains of the IgGs confirmed that the heavy chain is predominantly involved in binding to antigen. The light chain of porcine IgG contributes to the binding affinity toward an antigen and may function as a support platform for antibody stability. In summary, this study is the first to reveal the conserved antigenic profile of FMDV recognized by porcine B cells and provides a novel method for analysing the antibody response against FMDV in its natural hosts (i.e. pigs) at the clonal level.

Identification of Foot and Mouth Disease (FMD) Virus From Recently Outbreak Crossbred Cattle In Rajbari District of Bangladesh

Foot and mouth disease (FMD) is a devastating viral disease and endemic in nature in Bangladesh that causes huge economical losses. The present research work was aimed to determine the prevalence of FMD outbreaks and molecular detection of FMDV serotypes by uRT-PCR and gsRT-PCR test, respectively from crossbred cattle in the Rajbari district of Bangladesh during the period from January to June 2018. A total of 16 tongue epithelial samples were collected from clinically FMD suspected 2 to 3 years old crossbred cattle. 14 samples were positive by uRT-PCR. The detection rate of FMDV by uRT-PCR was 87.50%. Then uRT-PCR positive samples were serotype by gsRT-PCR. Serotype based prevalence of FMDV was 42.86%, 100%, 21.43% and 21.43% in O serotype, Asia-1 serotype, A serotype and mixed infection with Asia-1 and A, respectively. Considering the age, the prevalence of confirmed FMD outbreak was 42.86%, 35.71% and 21.43% at the age of 2, 2.5 and 3 years, respectively. Serotype A, O and Asia-1 is circulated in Rajbari district and required trivalent vaccine for prevention and control of FMD in that area. SAARC J. Agric., 19(1): 201-210 (2021)

Effective Diagnosis of Foot-And-Mouth Disease Virus (FMDV) Serotypes O and A Based on Optical and Electrochemical Dual-Modal Detection

Foot-and-mouth disease virus (FMDV) is a highly contagious disease that affects cloven-hoofed animals. The traditional diagnostic methods for FMDV have several drawbacks such as cross-reactivity, low sensitivity, and low selectivity. To overcome these drawbacks, we present an optical and electrochemical dual-modal approach for the specific detection of FMDV serotypes O and A by utilizing a magnetic nanoparticle labeling technique with resorufin β-d-glucopyranoside (res-β-glc) and β-glucosidase (β-glc), without the use of typical lateral flow assay or polymerase chain reaction. FMDV serotypes O and A were reacted with pan-FMDV antibodies that recognize all seven FMDV serotypes (O, A, C, Asia 1, SAT 1, SAT 2, and SAT 3). The antigen–antibody complex was then immobilized on magnetic nanoparticles and reacted with β-glc-conjugated FMDV type O or type A antibodies. Subsequently, the addition of res-β-glc resulted in the release of fluorescent resorufin and glucose owing to catalytic hydrolysis by β-glc. The detection limit of fluorescent signals using a fluorescence spectrophotometer was estimated to be log(6.7) and log(5.9) copies/mL for FMDV type O and A, respectively, while that of electrochemical signals using a glucometer was estimated to be log(6.9) and log(6.1) copies/mL for FMDV type O and A, respectively. Compared with a commercially available lateral flow assay diagnostic kit for immunochromatographic detection of FMDV type O and A, this dual-modal detection platform offers approximately four-fold greater sensitivity. This highly sensitive and accurate dual-modal detection method can be used for effective disease diagnosis and treatment, and will find application in the early-stage diagnosis of viral diseases and next-generation diagnostic platforms.

Characterising Foot-and-Mouth Disease Virus in Clinical Samples Using Nanopore Sequencing

The sequencing of viral genomes provides important data for the prevention and control of foot-and-mouth disease (FMD) outbreaks. Sequence data can be used for strain identification, outbreak tracing, and aiding the selection of the most appropriate vaccine for the circulating strains. At present, sequencing of FMD virus (FMDV) relies upon the time-consuming transport of samples to well-resourced laboratories. The Oxford Nanopore Technologies' MinION portable sequencer has the potential to allow sequencing in remote, decentralised laboratories closer to the outbreak location. In this study, we investigated the utility of the MinION to generate sequence data of sufficient quantity and quality for the characterisation of FMDV serotypes O, A, Asia 1. Prior to sequencing, a universal two-step RT-PCR was used to amplify parts of the 5′UTR, as well as the leader, capsid and parts of the 2A encoding regions of FMDV RNA extracted from three sample matrices: cell culture supernatant, tongue epithelial suspension and oral swabs. The resulting consensus sequences were compared with reference sequences generated on the Illumina MiSeq platform. Consensus sequences with an accuracy of 100% were achieved within 10 and 30 min from the start of the sequencing run when using RNA extracted from cell culture supernatants and tongue epithelial suspensions, respectively. In contrast, sequencing from swabs required up to 2.5 h. Together these results demonstrated that the MinION sequencer can be used to accurately and rapidly characterise serotypes A, O, and Asia 1 of FMDV using amplicons amplified from a variety of different sample matrices.

Comparative expression analysis of inflammatory and immune-related genes in cattle during acute infection with foot-and-mouth disease virus in Egypt

Introduction Foot-and-mouth disease is a highly infectious viral disease affecting all cloven-footed domestic animals. The three foot-and-mouth disease virus (FMDV) serotypes A, O and SAT2 are at present the greatest threat to susceptible animals in Egypt. The aim of the present study was, for the host factors associated with different FMDV infections in cattle during the acute phase, to compare these factors’ influence on the expression of the IL-10, TLR-2, TNF-α, CXCL10, CD48, NFATC4 and IFNG inflammatory and immune-related genes. Materials and methods Vesicular fluid and epithelium samples were obtained from at least three infected cattle on the same affected farm during three different FMDV outbreaks and were used for serotyping of the virus and for expression analysis of host genes. A two-step RT-PCR was used for diagnosis of the virus with primers specific for each serotype. Results In quantitative PCR analysis, the expression patterns of TLR-2 and IFNG were prominent, while NFATC4 expression was absent in all FMDV-infected cattle. The highest expression of CD48 was associated with increased expression of other inflammatory and immune-related genes (IL-10, TLR-2, TNF-α and IFNG), which may be an indication of rapid virus clearance. Conclusion The use of vesicular fluid and epithelium for investigation of viral and immune-related gene expression levels in acute FMDV infection is possible. Host-dependent variation in the expression of the studied genes was observed in different FMDV serotype outbreaks.

Differentiation of Foot and Mouth Disease Virus Serotypes in Animals in High-Risk Zones of Georgia

Foot and Mouth Disease (FMD) is the most important economic threat to the livestock industry. Outbreaks of FMD can have a devastating impact on livestock production and trade, resulting in significant economic losses in the agricultural sector. As a result, vaccination and containment programs have been implemented internationally to minimize the spread of FMDV. The national vaccination program has been implemented in Georgia since 2012, vaccinating Large Ruminants (LR) and Small Ruminants (SR) with trivalent (A, O, Asia1) vaccine twice annually. However, active seromonitoring surveillance still shows a high seroprevalence of the disease, indicating virus circulation. In this study we attempted to estimate the prevalence of different FMDV serotypes in various risk zones within Georgia. A total of 4991 small and large ruminants were tested for the presence of FMDV nonstructural proteins (NSP) in the blood, and the exact serotypes of positive animals were further investigated through structural protein (SP) based assays. The results show that significant percentages (6.6%) of vaccinated animals were affected by FMD, and those positive animals are usually affected by more than one FMDV serotype. As such, our data call upon a stricter vaccination and monitoring program for FMDV in Georgia, especially considering that due to the geographic location of Georgia, the presence of FMD can have significant impact on transit and can be a threat for other countries as well.

Isolation, Serotyping and Molecular Detection of Bovine FMD Virus from Outbreak Cases in Aba'ala District of Afar Region

Background: Among the top listed economically important transboundary livestock diseases of cattle, foot and mouth disease (FMD) is the leading bottleneck in livestock production and productivity in Ethiopia. On the basis of FMDV active outbreak cases, a cross sectional study was undertaken to collect samples from January, 2019 to March, 2020 intended for isolation, serotyping and molecular detection of FMDV in the study district. Purposive sampling method was applied to select the study area for the reason that the presence of current active FMD outbreak case report during the study period. Totally, 27 FMD suspected clinical samples were collected from clinically affected study population during field outbreak. Out of 27 samples, 18 of them were inoculated on cultured Baby hamster kidney (BHK-21) monolayer cells and all 27 samples were tested using conventional RT-PCR and sets of specific universal primers. Finally, the PCR products were visualized with UV illumination and imaged with gel documentation system. Results: The current study result revealed that out of 18 clinical samples subjected to virus isolation, 72.2%(n=13) of these cultures exhibited FMDV induced cytopathic effect (CPE) and the identified serotype was SAT-2 FMD virus. Out of 27 clinical samples tested by conventional RT-PCR, only 12 FMDV samples were found to be FMDV positive by universal primers. Out of 27 clinical samples detected by conventional reverse transcription polymerase chain reaction (RT-PCR), only 12 FMDV samples were found to be FMDV positive by universal primers.Conclusions: Our study finding indicated FMDV is prevalent in the study area and FMDV serotype SAT-2 was the causality for the outbreaks of the disease in the study area. Hence, region wise regular FMD outbreaks investigation, further phylogenetic analysis and vaccine matching field isolates should be carried out to know in depth data about FMDV serotypes and topotypes involving in afar region of Ethiopia for effective vaccine development and control of the disease.

Isolation, Serotyping and Molecular Detection of Bovine FMD Virus from Outbreak Cases in Aba'ala District of Afar Region

Background: Among the top listed economically important transboundary livestock diseases of cattle, foot and mouth disease (FMD) is the leading bottleneck in livestock production and productivity in Ethiopia. On the basis of FMDV active outbreak cases, a cross sectional study was undertaken to collect samples from January, 2019 to March, 2020 intended for isolation, serotyping and molecular detection of FMDV in the study district. Purposive sampling method was applied to select the study area for the reason that the presence of current active FMD outbreak case report during the study period. Totally, 27 FMD suspected clinical samples were collected from clinically affected study population during field outbreak. Out of 27 samples, 18 of them were inoculated on cultured Baby hamster kidney (BHK-21) monolayer cells and all 27 samples were tested using conventional RT-PCR and sets of specific universal primers. Finally, the PCR products were visualized with UV illumination and imaged with gel documentation system. Results: The current study result revealed that out of 18 clinical samples subjected to virus isolation, 72.2%(n=13) of these cultures exhibited FMDV induced cytopathic effect (CPE) and the identified serotype was SAT-2 FMD virus. Out of 27 clinical samples tested by conventional RT-PCR, only 12 FMDV samples were found to be FMDV positive by universal primers. Out of 27 clinical samples detected by conventional reverse transcription polymerase chain reaction (RT-PCR), only 12 FMDV samples were found to be FMDV positive by universal primers.Conclusions: Our study finding indicated FMDV is prevalent in the study area and FMDV serotype SAT-2 was the causality for the outbreaks of the disease in the study area. Hence, region wise regular FMD outbreaks investigation, further phylogenetic analysis and vaccine matching field isolates should be carried out to know in depth data about FMDV serotypes and topotypes involving in afar region of Ethiopia for effective vaccine development and control of the disease.