Foot-and-mouth Disease Virus Serotype Research Articles

Generation of monoclonal antibodies against foot-and-mouth disease virus SAT 2 and the development of a lateral flow strip test for virus detection.

Foot-and-mouth disease (FMD) remains a major economic concern for the livestock productivity in many developing countries and a continued threat to countries that are disease free because of its potential devastating impact on agricultural, food chain and tourism sectors. FMD virus (FMDV) is recognized as having seven serotypes: O, A, C, Asia 1, South African Territories (SAT) 1, 2, 3 and multiple subtypes within each serotype. FMD outbreaks due to SAT 2 have been reported in many African countries. The development of a rapid and easily performed test for FMD detection is critical for controlling FMD outbreaks and containing its spread. The present project developed a lateral flow immunochromatographic (LFI) strip test for the rapid detection of FMDV SAT 2. A panel of monoclonal antibodies (mAbs) against FMDV serotype SAT 2 was produced and characterized. One mAb (#10) was selected as the capture mAb because it reacted to all 23 SAT 2 isolates archived at the National Center for Foreign Animal Disease. The LFI strip test was developed using biotin-conjugated mAb #10, and the colloid gold-conjugated FMDV serotype-independent mAb as the detection mAb. A generic Rapid Assay Device (gRAD) with one test line and a control line was used for the test. The LFI strip test detected all 23 tested SAT 2 isolates and recent outbreak strains. The results indicated that the diagnostic specificity and sensitivity of the LFI strip test were greater than the double antibody sandwich (DAS) DAS ELISA. The ability of the LFI strip test to produce rapid diagnostic results will be useful for early on-site diagnosis during FMD outbreaks.

Investigating the temporal and spatial distribution of foot-and-mouth disease virus serotype C in the Region of South America, 1968-2016.

This study investigates the historical temporal trend and geographical distribution of the foot-and-mouth disease virus (FMDv) serotype C in South America; discussing the findings within the context of the actions and strategies carried out for the elimination of foot-and-mouth disease (FMD). This is the first time that such a comprehensive historical compilation has been carried out in the Region; hence, the study is intended as a reference and source of evidence about the presence/absence of FMDv serotype C in South America. Data on the occurrence of FMD were sourced from the Weekly Epidemiological Reports submitted by the countries to Pan AmericanFoot-and-Mouth DiseaseCenter(PANAFTOSA-PAHO/WHO) since 1972, and complemented with other sources of information from the 1968-1971 period. The temporal distribution was examined with local weighted regression (LOESS) to identify two temporal trends, that is, "smoothed" and "over-adjusted", utilising the time-series with the total number of cases per year, at Regional level. Thereafter the outbreaks were aggregated by decades and mapped by the first subnational administrative level. As a result, two major peaks of occurrence were identified, one in the 70s, with up to 1,193 outbreaks, and another in the 80s, with 380. Overall, the investigations show a clear regressive trend in the occurrence of serotype C, with a reduction in the number of outbreaks over-time, and with the subsequent reduction of affected locations. This study illustrates the contrast between the very limited presence over the last 20years - with only one event in 2004 - and the epidemic situation in the 1970s and 1980s, and suggests that serotype C of FMDv is no longer present in the Region.

Fully automated and integrated multiplex detection of high consequence livestock viral genomes on a microfluidic platform.

Differential diagnosis of diseases that share common clinical signs typically requires the performance of multiple independent diagnostic tests to confirm diagnosis. Diagnostic tests that can detect and discriminate between multiple differential pathogens in a single reaction may expedite, reduce costs, and streamline the diagnostic testing workflow. Livestock haemorrhagic diseases like classical swine fever (CSF), African swine fever (ASF), and vesicular diseases, such as foot-and-mouth disease (FMD), vesicular stomatitis (VS), and swine vesicular disease (SVD) can have an enormous impact on the livestock industry and economy of countries that were previously free of the diseases. Thus, rapid diagnosis of these diseases is critical for disease control. Here, we describe the development and initial laboratory validation of a novel fully automated user-developed assay for simultaneous detection and differentiation of multiple viruses of veterinary importance in a single reaction with minimal user-intervention. The user only performs sample loading, placement of consumables and reagents, selection and initiation of assay while all other processes (i.e., nucleic acid extraction, multiplex RT-PCR, reverse dot blot detection and result reporting) are performed fully automated. The current assay has a turn-around time of approximately 6hr and can simultaneously process up to 24 samples. The automated assay accurately and specifically detected 37 laboratory amplified strains of the five target viruses, including all seven serotypes of FMD virus, three genotypes of CSF virus, and two serotypes of VS virus. The assay also detected targeted viruses in a variety of clinical samples collected from infected animals, such as oral fluid, oral swab, nasal swab, whole blood, serum, as well as tonsil, spleen, kidney, and ileum. No cross-reactivity was observed with 15 nontarget viruses that affect livestock and samples from clinically healthy animals. To our knowledge, this is the first fully automated and integrated assay for simultaneous detection of multiple high consequence veterinary pathogens.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of swine leukocyte antigen 2 complexed with a CTL epitope AS64 derived from Asia1 serotype of foot-and-mouth disease virus

BackgroundCurrently, the structural characteristics of the swine major histocompatibility complex (MHC) class I molecule, also named swine leukocyte antigen class I (SLA-I) molecule need to be further clarified.ResultsA complex of SLA-I constituted by an SLA-2*HB01 molecule with swine β2-microglobulin and a cytotoxic T lymphocyte (CTL) epitope FMDV-AS64 (ALLRSATYY) derived from VP1 protein (residues 64–72) of Asia 1 serotype of foot-and-mouth disease virus (FMDV) was expressed, refolded, purified and crystallized. By preliminary X-ray diffraction analysis, it was shown that the diffraction resolution of the crystal was 2.4 Å and the space group belonged to P212121 with unit cell parameters a = 48.37, b = 97.75, c = 166.163 Å.ConclusionThis research will be in favor of illuminating the structural characteristics of an SLA-2 molecule associated with a CTL epitope derived from Asia1 serotype of FMDV.

Study on seroprevalence and serotyping of foot and mouth disease in Chad

Foot and mouth disease (FMD) is a highly contagious viral disease that affects all Artiodactyla. Seven immunologically distinct serotypes of FMD virus (FMDV) exist. In Chad, although FMD is included in the list of diseases monitored by the Chadian Animal Disease Surveillance Network (REPIMAT), the epidemiological situation remains unclear. A serological survey was conducted in the cattle population in eight of the nine administrative regions of the country (those regions with the highest cattle densities), to evaluate the prevalence and serotypes of circulating FMDV.A total of 796 sera from randomly selected cattle were analysed at the World Organisation for Animal Health/Food and Agriculture Organization of the United Nations FMD Reference Laboratory at the Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna(IZSLER), in Brescia (Italy). An enzyme-linked immunosorbent assay (ELISA), called 3ABC ELISA, was used to detect antibodies against non-structural proteins (NSPs), as well as a series of six competitive ELISAs to detect and serotype antibodies against the structural proteins of FMDV serotypes O, A, SAT 1, SAT 2, Asia 1 and C. Based on the detection of anti-NSP antibodies, the animal-level seroprevalence was 35.6%(95% confidence interval [CI]: 32.2–38.9) and the herd-level seroprevalence was 62.3% (95%CI: 53.0–71.5). FMD was present in all livestock administrative divisions surveyed, with a higher prevalence in southern regions, which are characterised by higher rainfall and humidity and more important transboundary animal movements. Cattle aged more than four years had a higher seroprevalence, which may be due to repeated exposure. Semi-sedentary farming and transhumance were also risk factors. Antibodies against serotypes A, O, SAT 1 and SAT 2 were detected.

Study on seroprevalence and serotyping of foot and mouth disease in Chad.

Foot and mouth disease (FMD) is a highly contagious viral disease that affects all Artiodactyla. Seven immunologically distinct serotypes of FMD virus (FMDV) exist. In Chad, although FMD is included in the list of diseases monitored by the Chadian Animal Disease Surveillance Network (REPIMAT), the epidemiological situation remains unclear. A serological survey was conducted in the cattle population in eight of the nine administrative regions of the country (those regions with the highest cattle densities), to evaluate the prevalence and serotypes of circulating FMDV.A total of 796 sera from randomly selected cattle were analysed at the World Organisation for Animal Health/Food and Agriculture Organization of the United Nations FMD Reference Laboratory at the Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna(IZSLER), in Brescia (Italy). An enzyme-linked immunosorbent assay (ELISA), called 3ABC ELISA, was used to detect antibodies against non-structural proteins (NSPs), as well as a series of six competitive ELISAs to detect and serotype antibodies against the structural proteins of FMDV serotypes O, A, SAT 1, SAT 2, Asia 1 and C. Based on the detection of anti-NSP antibodies, the animal-level seroprevalence was 35.6%(95% confidence interval [CI]: 32.2-38.9) and the herd-level seroprevalence was 62.3% (95%CI: 53.0-71.5). FMD was present in all livestock administrative divisions surveyed, with a higher prevalence in southern regions, which are characterised by higher rainfall and humidity and more important transboundary animal movements. Cattle aged more than four years had a higher seroprevalence, which may be due to repeated exposure. Semi-sedentary farming and transhumance were also risk factors. Antibodies against serotypes A, O, SAT 1 and SAT 2 were detected.

Development and evaluation of serotype-specific recombinase polymerase amplification combined with lateral flow dipstick assays for the diagnosis of foot-and-mouth disease virus serotype A, O and Asia1

BackgroundFoot-and-mouth disease (FMD) caused by foot-and-mouth disease virus (FMDV) is one of the most highly infectious diseases in livestock, and leads to huge economic losses. Early diagnosis and rapid differentiation of FMDV serotype is therefore integral to the prevention and control of FMD. In this study, a series of serotype-specific reverse transcription recombinase polymerase amplification assays combined with lateral flow dipstick (RPA-LFD) were establish to differentiate FMDV serotypes A, O or Asia 1, respectively.ResultsThe serotype-specific primers and probes of RPA-LFD were designed to target conserved regions of the FMDV VP1 gene sequence, and three primer and probe sets of serotype-specific RPA-LFD were selected for amplification of FMDV serotypes A, O or Asia 1, respectively. Following incubation at 38 °C for 20 min, the RPA amplification products could be visualized by LFD. Analytical sensitivity of the RPA assay was then determined with ten-fold serial dilutions of RNA of VP1 gene and the recombinant vector respectively containing VP1 gene from FMDV serotypes A, O or Asia1, the detection limits of these assays were 3 copies of plasmid DNA or 50 copies of viral RNA per reaction. Moreover, the specificity of the assay was assessed, and there was no cross reactions with other viruses leading to bovine vesicular lesions. Furthermore, 126 clinical samples were respectively detected with RPA-LFD and real-time PCR (rPCR), there was 98.41% concordance between the two assays, and two samples were positive by RPA-LFD but negative in rPCR, these were confirmed as FMDV-positive through viral isolation in BHK-21 cells. It showed that RPA-LFD assay was more sensitive than the rPCR method in this study.ConclusionThe development of serotype-specific RPA-LFD assay provides a rapid, sensitive, and specific method for differentiation of FMDV serotype A, O or Asia1, respectively. It is possible that the serotype-specific RPA-LFD assay may be used as a integral protocol for field detection of FMDV.

Molecular epidemiology and phylogenetic analysis of foot and mouth disease virus type ‘O’ and evaluation of its carrier state in cattle of Assam by Real time PCR

The molecular epidemiological study of foot-and-mouth disease virus (FMDV) has been carried out from different outbreaks in Assam the present study is based on the nucleotide sequencingof circulating FMDV serotype. The samples were subjected to sandwich ELISA, multiplex-PCR and molecular phylogeny to identify the type species. The phylogenetic analysis of virus sequence revealed similarity with theBangladesh isolates in the major branching pattern. The serotype ‘O’has found to be dominant and responsible for most of the recentoutbreaks.Thepersistence of serotype ‘O’ and cytokines expression of IL-1á, IL-1â, IFN-á, TNF-á in blood of recovered animals were done by Real time PCR. The findings indicated that IL-1á, IFN-á and TNF-á genes were up-regulated upto 3 months post infection but IL-1â found to be down regulated with progression of recovery. The present study thus supports that real-time PCR is a powerful technique for reliable detection of persistent FMDV in recovered animals.

Serotyping of foot-and-mouth disease virus using oxford nanopore sequencing

Foot-and-mouth disease virus (FMDV), belonging to the family of Picornaviridae, infects mostly cloven-hoofed animals and leads to huge economic losses. Since there is no cross-protection between the seven serotypes of FMDV, effective vaccination relies on the knowledge of the serotype causing the outbreak. The most common methods of serotyping are antigen ELISAs and amplification-based sequencing. Serotype-specific PCR methods exist but have limitations due to emerging mutants within serotypes. Sequencing is a promising technology, but currently suffers from cumbersome procedures and long turnaround times. In this study, we have established a novel sequencing protocol relying on nanopore sequencing and offline BLAST search. The procedure was completed in 5 h including RNA extraction, reverse transcription, second-strand synthesis, barcoding, sequencing and data analysis, which did not require a bioinformatician. In total, 12,193 sequence files were obtained. The offline BLAST search to the P1 region revealed the most successful categorization of the seven FMDV serotypes (specificity: 98.3%) over whole genome (24.8%), P2 (23.6%) and P3 (21.4%). In conclusion, our protocol enables rapid and reliable FMDV serotyping. The whole procedure can be conducted with a mobile suitcase laboratory, which is easy to use at the point of need in endemic countries.

Versatility of the adenovirus-vectored foot-and-mouth disease vaccine platform across multiple foot-and-mouth disease virus serotypes and topotypes using a vaccine dose representative of the AdtA24 conditionally licensed vaccine

We investigated the serotype- and topotype versatility of a replication-deficient human adenovirus serotype 5 vectored foot-and-mouth disease (FMD) vaccine platform (AdtFMD). Sixteen AdtFMD recombinant subunit monovalent vaccines targeting twelve distinct FMD virus (FMDV) serotype/topotypes in FMD Regional Pools I-VII were constructed. The AdtA24 serotype conditionally licensed vaccine served as the basis for vaccine design and target dose for cattle clinical trials. Several vaccines contained an additional RGD motif genetic insertion in the adenovector fiber knob, and/or a full-length 2B gene insertion in the FMDV P1 gene cassette. In 13 of the 22 efficacy studies conducted, naïve control and AdtFMD vaccinated cattle were challenged intradermolingually at 2 weeks post-vaccination using a FMDV strain homologous to the AdtFMD vaccine strain. Each of the 16 AdtFMD vaccines were immunogenic based on the presence of homologous neutralizing antibodies in the serum of approximately 90% of total vaccinates (n = 375) on the day of challenge. Importantly, for 75% of vaccines tested, the effective dose that conferred 100% protection against clinical FMD was identical to or in some cases lower than, the minimum protective dose for the conditionally licensed AdtA24 vaccine formulated with ENABL® adjuvant. Results also confirmed the capability of the AdtFMD vaccine platform to differentiate infected from vaccinated animals (DIVA) across the five FMDV serotypes evaluated. Collectively, this comprehensive set of FMD cattle vaccine dose ranging studies highlights the serotype- and topotype versatility of the AdtFMD vaccine platform for further development, licensure, and application in FMD outbreak control and disease eradication efforts.

Indirect ELISA using a multi-epitope recombinant protein to detect antibodies against foot-and-mouth disease virus serotype O in pigs

Foot-and-mouth disease (FMD) is a devastating animal disease. A previously developed multi-epitope protein B4 vaccine of the FMD virus (FMDV) serotype O provides safety advantages over inactivated vaccines and could be used to prevent and control FMD in pigs. Commercial enzyme-linked immunosorbent assay (ELISA) kits for assessing vaccine efficacy are available for the inactivated vaccines but not for the multi-epitope protein vaccine. In this study, multi-epitope protein B4 was expressed in Escherichia coli, and an indirect ELISA (I-ELISA) was developed to detect antibodies against FMDV serotype O in pigs. The specificity and sensitivity were 96.7% and 95.9%, respectively. B4-vaccinated pigs yielded B4 I-ELISA serum values that were positively correlated with clinical protection against challenge with FMDV serotype O. The I-ELISA’s ability to detect antibodies from animals vaccinated with the inactivated vaccine was also evaluated, and the B4 I-ELISA values were significantly positively correlated with liquid-phase blocking ELISA (LPBE) titers (r = 0.6708, p < 0.0001); thus, the I-ELISA was also suitable for detection of antibodies from swine vaccinated with the inactivated vaccine.

Recombination of host cell mRNA with the Asia 1 foot-and-mouth disease virus genome in cell suspension culture.

Foot-and-mouth disease virus (FMDV) exhibits high mutation rates during replication. In this study, an isolate of FMDV serotype Asia-1 was serially passaged in a BHK-21 cell monolayer and then adapted to serum-free BHK-21 cell suspension culture to produce a seed virus for production of an inactivated vaccine. Analysis of the sequence encoding the structural proteins of the virus at various passages showed the presence of overlapping peaks in sequencing electropherograms after nucleotide 619 of VP1 in viruses recovered from the fourth passage in suspension culture, suggesting the possible introduction of an insertion or deletion into this portion of the viral genome of our seed virus stock. To evaluate this phenomenon, a virus designated "Vac-Asia1-VDLV", was isolated by plaque purification from the tenth passage in suspension culture. Sequencing results showed that a 12-nt-long exogenous sequence was inserted into the 3' end of the VP1 coding region at the position where the original overlapping peaks were identified. Analysis of the host cell transcriptome showed that the 12-nt sequence was identical to a highly expressed sequence in BHK-21 cells, strongly suggesting that recombination between the FMDV genome and host cell mRNA produced the recombinant virus. A growth curve showed that the virus with the 12-nt insertion reached a peak earlier than the parental strain and that this virus had acquired the ability to bind to the cell surface by a mechanism that was not dependent on integrin or the heparan sulfate receptor. This novel pathogen-host cell recombination event is discussed in terms of the mechanism of viral RNA replication and the phenotypic constraints of FMDV biology and evolution.

Outbreak investigations of foot and mouth disease virus in Nepal between 2010 and 2015 in the context of historical serotype occurrence.

Foot and Mouth Disease (FMD) is endemic in Nepal and causes substantial economic losses in the livestock industry. The goal of this study was to perform an epidemiological analysis of FMD outbreaks reported to the Veterinary Epidemiology Center, Tripureshwor, Nepal during 2010–2015, in order to strengthen the National FMD Control Program. These current data were considered in the context of historical data on FMD virus (FMDV) serotypes detected in the country between 1965 and 2015. During 2010–2015, a total of 1333 livestock holdings reported FMD outbreaks in Nepal. On average, 71.2 animals were affected in each outbreak, with a case fatality rate of 3.6%. FMD was reported throughout the country, and the proportion of affected holdings was not significantly among eco‐zones, regions, or species. The Hill eco‐zone had the highest number of holdings affected (782), followed by Mountain (304), and Terai (247). When analysed by the developmental region, the Western (381) and Central (368) Developmental Regions had the highest numbers of holdings affected. Cattle were the most frequently affected species (39%), followed by buffalo (33%), and goats (19%). FMD occurred throughout the year, with peaks in winter (December/January) and in the pre‐monsoon period (April/May). Between 1965 and 2015 FMDV serotype O had the highest prevalence (81%), followed by Asia‐1 (11%), A (6%), and C (2%). Serotype C was not detected after 1996, and only serotype O was reported after 2011. These descriptive analyses provide critical landmarks to establish baselines, and document early progress of the ongoing Progressive Control Pathway of FMD (PCP‐FMD) which could be useful in Nepal and other South Asian nations.

Visual and equipment-free reverse transcription recombinase polymerase amplification method for rapid detection of foot-and-mouth disease virus

BackgroundFoot-and-mouth disease (FMD), which is caused by foot-and-mouth disease virus (FMDV), is a highly contagious tansboundary disease of cloven-hoofed animals and causes devastating economic damages. Accurate, rapid and simple detection of FMDV is critical to containing an FMD outbreak. Recombinase polymerase amplification (RPA) has been explored for detection of diverse pathogens because of its accuracy, rapidness and simplicity. A visible and equipment-free reverse-transcription recombinase polymerase amplification assay combined with lateral flow strip (LFS RT-RPA) was developed to detect the FMDV using primers and LF probe specific for the 3D gene.ResultsThe FMDV LFS RT-RPA assay was performed successfully in a closed fist using body heat for 15 min, and the products were visible on the LFS inspected by the naked eyes within 2 min. The assay could detect FMDV serotypes O, A and Asia1, and there were no cross-reactions with vesicular stomatitis virus (VSV), encephalomyocarditis virus (EMCV), classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus 2 (PCV2) and pseudorabies virus (PRV). The analytical sensitivity was 1.0 × 102 copies in vitro transcribed FMDV RNA per reaction, which was the same as a real-time RT-PCR. For the 55 samples, FMDV RNA positive rate was 45.5% (25/55) by LFS RT-RPA and 52.7% (29/55) by real-time RT-PCR. For the LFS RT-RPA assay, the positive and negative predicative values were 100% and 80%, respectively.ConclusionsThe performance of the LFS RT-RPA assay was comparable to real-time RT-PCR, while the LFS RT-RPA assay was much faster and easier to be performed. The developed FMDV LFS RT-RPA assay provides an attractive and promising tool for rapid and reliable detection of FMDV in under-equipped laboratory and at point-of-need facility, which is of great significance in FMD control in low resource settings.

Mutation in the VP2 gene of P1-2A capsid protein increases the thermostability of virus-like particles of foot-and-mouth disease virus serotype O.

Foot-and-mouth disease (FMD) is an economically important, global disease of cloven-hoofed animals. The conventional vaccine could bring down the incidence of disease in many parts of the world but has many limitations and in India, the disease is enzootic. More promisingly, the alternate vaccine candidates, virus-like particles (VLPs) are as immunogenic as a native virus but are more labile to heat than the live virus capsids. To produce stable VLPs, a single amino acid residue was mutated at 93 and 98 positions at VP2 inter-pentamer region of the P1-2A gene of FMD virus serotype O (IND/R2/75). The mutated capsid protein was expressed in insect cells and characterized for temperature and varying pH stability. Out of S93Y, S93F, S93C, S93H, and Y98F mutant, VLPs, S93Y, S93F, and Y98F showed improved stability at 37°C for 75days compared to wild capsid, which was evaluated by sandwich ELISA. Further, the stability analysis of purified VLPs either by differential scanning fluorescence (DSF) stability assay at different temperatures and pH conditions or by dissociation kinetics showed that the Y98F mutant VLPs were more stable than S93Y, S93F, S93C, and S93H mutant and wild-type VLPs. Immunization of guinea pigs with Y98F VLPs induced neutralizing antibodies and 60% of the animals were protected from the FMDV "O" 100 GPID50 challenge virus.

Universal detection of foot and mouth disease virus based on the conserved VP0 protein

Background: Foot and mouth disease virus (FMDV), a member of the picornaviridae that causes vesicular disease in ungulates, has seven serotypes and a large number of strains, making universal detection challenging. The mature virion is made up of 4 structural proteins, virus protein (VP) 1 – VP4, VP1-VP3 of which form the outer surface of the particle and VP4 largely contained within. Prior to mature virion formation VP2 and VP4 occur together as VP0, a structural component of the pre-capsid which, as a result of containing the internal VP4 sequence, is relatively conserved among all strains and serotypes. Detection of VP0 might therefore represent a universal virus marker. Methods: FMDV virus protein 0 (VP0) was expressed in bacteria as a SUMO fusion protein and the SUMO carrier removed by site specific proteolysis. Rabbit polyvalent sera were generated to the isolated VP0 protein and their reactivity characterised by a number of immunoassays and by epitope mapping on peptide arrays. Results: The specific VP0 serum recognised a variety of FMDV serotypes, as virus and as virus-like-particles, by a variety of assay formats. Epitope mapping showed the predominant epitopes to occur within the unstructured but highly conserved region of the sequence shared among many serotypes. When immunogold stained VLPs were assessed by TEM analysis they revealed exposure of epitopes on the surface of some particles, consistent with particle breathing hitherto reported for some other picornaviruses but not for FMDV. Conclusion: A polyvalent serum based on the VP0 protein of FMDV represents a broadly reactive reagent capable of detection of many if not all FMDV isolates. The suggestion of particle breathing obtained with this serum suggests a reconsideration of the FMDV entry mechanism.

Animal-level risk factors associated with foot-and-mouth disease in cattle and buffalo in Egypt

There is a paucity of the investigation of risk factors associated with foot-and-mouth disease (FMD) in Egypt. Subsequently, in the present study the impact of risk factors associated with FMD infection was analyzed in cattle and buffalo reared in Qalyubia governorate, Egypt. Serum samples were collected from 288 animals (n = 90 water buffalo (Bubalus bubalis) and n = 198 cattle) and adapted for detection of FMD virus infection by ELISA assay with monoclonal antibody (MELISA) to type serotype O. Direct FMD virus serotype was detected in 115 animals (n = 82 cattle and n = 33 buffalo). Univariate analysis revealed a significant association between FMD infection and the fattening animals (P = 0.024; odds ratio (OR) 2.118; confidence interval (CI) 1.105–4.058). Multivariate statistics demonstrated that locality was the potential risk factors for infection by FMD virus (P = 0.001; OR 31.613; CI 13.791–72.466). The present investigation draws attention and gives the impression to the potential risk factors associated with FMD-infected cases in cattle and buffalo in Qalyubia governorate in Egypt. Recognition of the potential risk factors associated with FMD-infected cases offer a novel approach to construct the best preventive measures.

An improved, rapid competitive ELISA using a novel conserved 3B epitope for the detection of serum antibodies to foot-and-mouth disease virus.

The highly contagious foot-and-mouth disease virus (FMDV) afflicts cloven-hoofed animals, resulting in significant costs because of loss of trade and recovery from disease. We developed a sensitive, specific, and rapid competitive ELISA (cELISA) to detect serum antibodies to FMDV. The cELISA utilized a monoclonal blocking antibody specific for a highly conserved FMDV nonstructural 3B epitope, a recombinant mutant FMDV 3ABC coating protein, and optimized format variables including serum incubation for 90 min at 20–25°C. Samples from 16 animals experimentally infected with one FMDV serotype (A, O, Asia, or SAT-1) demonstrated early detection capacity beginning 7 d post-inoculation. All samples from 55 vesicular stomatitis virus antibody-positive cattle and 44 samples from cloven-hoofed animals affected by non-FMD vesicular diseases were negative in the cELISA, demonstrating 100% analytical specificity. The diagnostic sensitivity was 100% against sera from 128 cattle infected with isolates of all FMDV serotypes, emphasizing serotype-agnostic results. Diagnostic specificities of U.S. cattle (n = 1135) and swine (n = 207) sera were 99.4% and 100%, respectively. High repeatability and reproducibility were demonstrated with 3.1% coefficient of variation in percent inhibition data and 100% agreement using 2 kit lots and 400 negative control serum samples, with no difference between bench and biosafety cabinet operation. Negative results from vaccinated, uninfected cattle, pig, and sheep sera confirmed the DIVA (differentiate infected from vaccinated animals) capability. This rapid (<3 h), select agent–free assay with high sensitivity and specificity, DIVA capability, and room temperature processing capability will serve as a useful tool in FMDV surveillance, emergency preparedness, response, and outbreak recovery programs.

Biological Assay of Foot and Mouth Disease Virus (FMDV) Serotypes for Titrating BLRI Developed Trivalent FMD Vaccines Seed

Foot and Mouth Disease (FMD) is an important viral as well as transboundary disease affecting almost all cloven-hoofed animals. The aim of the present study is the focused on determination of biological titer by tissue culture infective dose50 (TCID50) assay of currently available FMD virus serotype in Bangladesh. For adaptation of FMD virus (FMDV), BHK-21 cell line was used. BHK-21 cell subculture was done from preserved cultured bottle of Foot and Mouth Disease (FMD) Research Laboratory of Bangladesh Livestock Research Institute (BLRI), Savar, Dhaka. RT-PCR confirmed selected three different positive serotypes (O, A and Asia 1) of FMD virus were inoculated into BHK-21 cell culture and cytopathic effects (CPE) were observed after adaptation into BHK-21 cell from 3rd to 5th passages. Clear infectious BHK-21 cell culture fluid was collected and preserved at -80℃ temperature. The TCID50 assay was performed to determine the biological titer of the three positive serotype of FMDV. The biological titer of this study was found 106.5/ml viral titer for O type, 106.75/ml viral titer for A type, 106.66/ml viral titer for Asia-1 type. These three specific serotypes can be used as vaccine seed against FMD virus. The effective vaccination of susceptible animals is considered to be the corner stone to the disease in our country Bangladesh. The findings of this study can be helpful for the trivalent vaccine development in Bangladesh and it may effective in limiting the spread of FMD.

Using size-exclusion chromatography to quantify the 146S antigen in inactivated foot-and-mouth disease vaccine

The aim of this study is to quantify the 146S antigen in foot-and-mouth disease virus (FMDV) inactivated vaccine by size-exclusion chromatography (SEC). The analysis was performed on a TSKgel G4000SWXL column (7.8 mm×30 cm), with a pH 7.2 buffer salt system as the mobile phase. The flow rate was 0.6 mL/min, the injection volume was 100 μL and the detection wavelength was 259 nm. The calibration curve was established by using purified inactivated FMDV (serotype O) 146S antigen; 3 batches of vaccine formulated by inactivated antigen solution were tested to verify the accuracy, reproducibility, specificity and tolerability of the method. At last 16 batches of vaccine were determined by the SEC method. Results showed a good linearity between peak area and concentration of 146S antigen in the range between 0.56 and 67.42 μg/mL (R2=0.996, n=10), and the average recovery rate of 146S antigen in the 3 batches of vaccine formulated in lab were 93.6% (RSD=2.7%, n=3), 102.3% (RSD=2.6%, n=3), and 95.5% (RSD=5.1%, n=3). The method was proved accurate and reliable with good reproducibility (RSD=0.5%, n=6), and applied to determine 16 batches of the commercial FMDV vaccine. According to the above results, the SEC method is high effective for 146S antigen quantify in the inactivated FMDV vaccine and would provide strong support for the vaccine quality control.