Reiko Yamazoe

Foot-and-Mouth Disease Virus Antigen Detection Enzyme-Linked Immunosorbent Assay Using Multiserotype-Reactive Monoclonal Antibodies

ABSTRACT Monoclonal antibody (MAb)-based sandwich direct enzyme-linked immunosorbent assay (MSD-ELISA) methods that can detect foot-and-mouth disease virus (FMDV) antigens, both multiserotype (MSD-ELISA/MS) (for O, A, C, and Asia 1) and single-serotype (MSD-ELISA/SS) (for O, A, and Asia 1, specifically), were developed. MAb 1H5 was used as an antigen-trapping antibody that reacted with all seven serotypes of FMDV. The MAbs 71F2, 70C4, 16C6, and 7C2 were used as peroxidase-labeled detecting antibodies for multiple serotypes (O, A, C, and Asia 1), type O, type A, and type Asia 1, respectively, in both MSD-ELISA/MS and MSD-ELISA/SS. Our MSD-ELISAs showed high specificity. They produced a very low background of negative samples (buffer, plasma, and saliva) and were able to detect FMDV antigens from clinical samples (plasma and saliva), with results correlating with those of real-time reverse transcription-PCR. In terms of sensitivity, the MSD-ELISAs showed higher optical density values against each diluted serotype antigen than the indirect sandwich ELISA method, which is currently recommended in the manual of the World Organization for Animal Health. The sensitivity and specificity of the MSD-ELISAs seem to be sufficient for the antigenic diagnosis of FMDV.

Dose-dependent responses of pigs infected with foot-and-mouth disease virus O/JPN/2010 by the intranasal and intraoral routes

Foot-and-mouth disease virus (FMDV) infection was successfully initiated in pigs by intraoral inoculation of both 106 and 103 TCID50 of FMDV O/JPN/2010 isolated from the 2010 epidemic in Japan. By intranasal inoculation, infection was established in pigs with 106 TCID50 of the isolate, but not with 103 TCID50 of the isolate. In the pigs inoculated with 106 TCID50 of the isolate, viruses and viral RNAs were obtained earlier from the pigs inoculated by the intraoral route than from the pigs inoculated by the intranasal route. These results support the theory that primary infection of a pig herd is more likely to occur by ingestion than by inhalation and that the oral cavity is likely to be a major entry route for FMDV in naturally exposed pigs.

Experimental infection of cattle and goats with a foot-and-mouth disease virus isolate from the 2010 epidemic in Japan.

In this study, we carried out experimental infections in cattle and goats using a foot-and-mouth disease virus (FMDV) isolate from the 2010 epidemic in Japan to analyze clinical manifestations, virus-shedding patterns and antibody responses in the animals. We found that the FMDV O/JPN/2010 isolate is virulent in cattle and goats, produces clinical signs, is spread efficiently by direct contact within the same species, and is persistently infectious in cattle. Quantitative analysis of levels of viral RNA in the tissues of cattle and goats infected with the isolate showed that the pharyngeal region is an important major target of the FMDV O/JPN/2010. Time course data of viral loads, excretion and transmission of the FMDV O/JPN/2010 in this study are key in providing quantitative data essential for epidemiological investigation and risk analysis in relation to disease controls.

Comparative performance of fetal goat tongue cell line ZZ-R 127 and fetal porcine kidney cell line LFBK-αvβ6 for Foot-and-mouth disease virus isolation

The fetal goat tongue cell line ZZ-R 127 and the fetal porcine kidney cell line LFBK-αvβ6 have been reported to have high sensitivity to various Foot-and-mouth disease virus (FMDV) strains. The suitability of ZZ-R 127 cells for FMDV isolation not only from epithelial suspensions but also from other clinical samples has already been confirmed in a previous study. However, to our knowledge, the suitability of LFBK-αvβ6 cells has not been evaluated using clinical samples other than epithelial materials. In addition, both cell lines have never been compared, in terms of use for FMDV isolation, under the same conditions. Therefore, in the current study, the virus isolation rates of both cell lines were compared using clinical samples collected from animals infected experimentally with FMDV. Viruses were successfully isolated from clinical samples other than epithelial suspensions for both cell lines. The virus isolation rates for the 2 cell lines were not significantly different. The Cohen kappa coefficients between the virus isolation results for both cell lines were significantly high. Taken together, these results confirmed the suitability of LFBK-αvβ6 cells for FMDV isolation from clinical samples other than epithelial suspensions. The levels of susceptibility of both cell lines to FMDV isolation were also confirmed to be almost the same.

Development and Evaluation of a Rapid Antigen Detection and Serotyping Lateral Flow Antigen Detection System for Foot-and-Mouth Disease Virus.

We developed a lateral flow strip using monoclonal antibodies (MAbs) which allows for rapid antigen detection and serotyping of foot-and-mouth disease virus (FMDV). This FMDV serotyping strip was able to detect all 7 serotypes and distinguish serotypes O, A, C and Asia1. Its sensitivities ranged from 103 to 104 of a 50% tissue culture infectious dose of each FMDV stain; this is equal to those of the commercial product Svanodip (Boehringer Ingelheim Svanova, Uppsala, Sweden), which can detect all seven serotypes of FMDV, but does not distinguish them. Our evaluation of the FMDV serotyping strip using a total of 118 clinical samples (vesicular fluids, vesicular epithelial emulsions and oral and/or nasal swabs) showed highly sensitive antigen detection and accuracy in serotyping in accordance with ELISA or RT-PCR. To the best of our knowledge, this is the first report on any FMDV serotyping strip that provides both rapid antigen detection and serotyping of FMDV at the same time on one strip without extra devices. This method will be useful in both FMD-free countries and FMD-infected countries, especially where laboratory diagnosis cannot be carried out.

Genetic diversity of RNA segments 5, 7 and 9 of the Palyam serogroup orbiviruses from Japan, Australia and Zimbabwe ☆

Reverse transcriptase-polymerase chain reaction (RT-PCR) methods, based on the sequences of RNA segments 5, 7 and 9 of Chuzan virus, were established for specific detection and molecular characterization of the Palyam serogroup orbiviruses. Nucleotide sequences obtained from the amplified cDNA fragments of these three genes of 24 isolates were analyzed and compared individually to determine the intra-serogroup phylogenetic relationship of Japanese, Australian and Zimbabwean isolates. It seems that Chuzan virus isolates in Japan are genetically stable. Interestingly, mutations have occurred almost simultaneously on these three genes of Chuzan virus. In all cases, isolates from the same geographical area were closely related to each other at the molecular level, irrespective of serotype. The data suggested that the Palyam serogroup viruses can be differentiated into geographically distinct groups and that the viruses evolve independently in the different gene pools. A strain KY-115 was considered to be produced by reassortment of genome segments between different groups. Restriction fragment length polymorphism (RFLP) analysis of these PCR products is useful for rapid discrimination of isolates and for detection of genetic mutations.

Experimental infections using the foot-and-mouth disease virus O/JPN/2010 in animals administered a vaccine preserved for emergency use in Japan

The effectiveness of a vaccine preserved for emergency use in Japan was analyzed under experimental conditions using cows and pigs in order to retrospectively evaluate the effectiveness of the emergency vaccination performed in the 2010 epidemic in Japan. Cows and pigs were administered a vaccine preserved for emergency use in Japan at 3 or 30 days before virus infection (dbv) and were subsequently infected with the foot-and-mouth disease virus (FMDV) O/JPN/2010, which was isolated in the 2010 epidemic in Japan. All animals vaccinated at 30 dbv and one of three pigs vaccinated at 3 dbv showed no vesicular lesions during the experimental period. The virus titers and viral RNA loads obtained from clinical samples were lower in the vaccinated cows than in the non-vaccinated cows. The viral excretion periods were shorter in the vaccinated cows than in the non-vaccinated cows. In contrast, in the vaccinated pigs, the virus titers and viral RNA loads obtained from the samples, except for those obtained from sera, were not decreased significantly, and the viral excretion periods were not sufficiently shortened. These results suggest that the vaccine can protect against clinical signs of infection by the FMDV O/JPN/2010 in animals; however, it should be noted that in vaccinated and infected animals, especially pigs, clinical samples, such as saliva and nasal swabs, may contain excreted viruses, even if no clinical signs were exhibited.

Further evaluation of an ELISA kit for detection of antibodies to a nonstructural protein of foot-and-mouth disease virus

An ELISA kit for detection of antibodies to a nonstructural protein of foot-and-mouth disease (FMDV) was further evaluated using sequentially collected serum samples of experimentally infected animals, because the sensitivity of the kit used in a previous study was significantly low in field animals. The kit fully detected antibodies in infected animals without vaccination; however, the first detections of antibodies by the kit were later than those by the liquid-phase blocking ELISA that is used for serological surveillance in the aftermath of outbreaks in Japan, for detection of antibodies to structural proteins of FMDV. Additionally, although the kit effectively detected antibodies in infected cattle with vaccination, there were several infected pigs with vaccination for which the kit did not detect antibodies during the experimental period. Taken together, the kit may not be suitable for serological surveillance after an FMD outbreak either with or without emergency vaccination in FMD-free countries.

Availability of a fetal goat tongue cell line ZZ-R 127 for isolation of Foot-and-mouth disease virus (FMDV) from clinical samples collected from animals experimentally infected with FMDV

The availability of the fetal goat tongue cell line ZZ-R 127 for the isolation of Foot-and-mouth disease virus (FMDV) has not been evaluated using clinical samples other than epithelial suspensions. Therefore, in the current study, the availability of ZZ-R 127 cells for the isolation of FMDV was evaluated using clinical samples (e.g., sera, nasal swabs, saliva, feces, and oropharyngeal fluids) collected from animals experimentally infected with an FMDV isolate. Virus isolation rates for the ZZ-R 127 cells were statistically higher than those for the porcine kidney cell line (IB-RS-2) in experimental infections using cattle, goats, and pigs (P < 0.01). Virus titers in the ZZ-R 127 cells were also statistically higher than those in the IB-RS-2 cells. The availability of ZZ-R 127 cells for the isolation of FMDV not only from epithelial suspensions but also from other clinical samples was confirmed in the current study.

Neutralizing monoclonal antibody sandwich liquid-phase blocking enzyme-linked immunosorbent assay for detection of Foot-and-mouth disease virus type O antibodies.

Liquid-phase blocking enzyme-linked immunosorbent assay (LPBE) using the neutralizing monoclonal antibody (mAb) sandwich method (M-LPBE) for detection of Foot-and-mouth disease virus (FMDV) type O antibodies was developed. Two neutralizing mAbs, 72C1 and 65H6, were raised against the FMDV O/JPN/2000 strain, and used as trapping and peroxidase-labeled detecting antibodies, respectively. Sera from animals experimentally infected with FMDV showed specific positive results by M-LPBE, which were correlated with the results of the virus neutralization test (VNT). When 303 negative bovine and 302 negative swine sera were tested, the specificity of M-LPBE was 100% and 99.7%, respectively. In addition, nine samples that had been collected in 2000 in Japan and regarded as evidently false positives by LPBE (supplied by the World Reference Laboratory for Foot-and-Mouth Disease) were uniformly negative by M-LPBE, just like VNT. Therefore, M-LPBE seems to have sufficient specificity for FMDV type O antibody screening and diagnosis.