Bernd Haas

A Portable Reverse Transcription Recombinase Polymerase Amplification Assay for Rapid Detection of Foot-and-Mouth Disease Virus

Foot-and-mouth disease (FMD) is a trans-boundary viral disease of livestock, which causes huge economic losses and constitutes a serious infectious threat for livestock farming worldwide. Early diagnosis of FMD helps to diminish its impact by adequate outbreak management. In this study, we describe the development of a real-time reverse transcription recombinase polymerase amplification (RT-RPA) assay for the detection of FMD virus (FMDV). The FMDV RT-RPA design targeted the 3D gene of FMDV and a 260 nt molecular RNA standard was used for assay validation. The RT-RPA assay was fast (4–10 minutes) and the analytical sensitivity was determined at 1436 RNA molecules detected by probit regression analysis. The FMDV RT-RPA assay detected RNA prepared from all seven FMDV serotypes but did not detect classical swine fever virus or swine vesicular disease virus. The FMDV RT-RPA assay was used in the field during the recent FMD outbreak in Egypt. In clinical samples, reverse transcription polymerase chain reaction (RT-PCR) and RT-RPA showed a diagnostic sensitivity of 100% and 98%, respectively. In conclusion, FMDV RT-RPA was quicker and much easier to handle in the field than real-time RT-PCR. Thus RT-RPA could be easily implemented to perform diagnostics at quarantine stations or farms for rapid spot-of-infection detection.

High potency vaccines induce protection against heterologous challenge with foot-and-mouth disease virus

In a series of three homologous and eight heterologous challenge experiments, it was shown that high potency vaccines against foot-and-mouth disease (FMD) serotype A can induce protection even against heterologous challenge infection with viruses that give low r-values with the vaccine strains. The challenge virus specific neutralizing antibody response on the day of challenge (21 days post vaccination) generally correlated with protection.

Highly Sensitive Fetal Goat Tongue Cell Line for Detection and Isolation of Foot-and-Mouth Disease Virus

ABSTRACT A fetal goat cell line (ZZ-R 127) supplied by the Collection of Cell Lines in Veterinary Medicine of the Friedrich Loeffler Institute was examined for susceptibility to infection by foot-and-mouth disease (FMD) virus (FMDV) and by two other viruses causing clinically indistinguishable vesicular conditions, namely, the viruses of swine vesicular disease and vesicular stomatitis. Primary bovine thyroid (BTY) cells are generally the most sensitive cell culture system for FMDV detection but are problematic to produce, particularly for laboratories that infrequently perform FMD diagnostic tests and for those in countries where FMD is endemic that face problems in sourcing thyroid glands from FMD-negative calves. Strains representing all seven serotypes of FMDV could be isolated in ZZ-R 127 cells with a sensitivity that was considerably higher than that of established cell lines and within 0.5 log of that for BTY cells. The ZZ-R 127 cell line was found to be a sensitive, rapid, and convenient tool for the isolation of FMDV and a useful alternative to BTY cells for FMD diagnosis.

Comparison of the plaque test and reverse transcription nested PCR for the detection of FMDV in nasal swabs and probang samples

In order to compare the sensitivity of assays for the diagnosis of foot-and mouth disease (FMD), a cell suspension plaque test on BHK21-CT cells and a reverse transcription nested PCR (RT-nPCR) were used to examine 485 nasal swabs and 227 probang samples obtained from FMDV-infected cattle during vaccine potency tests. In nasal swabs, FMDV could be detected by both tests before the onset of clinical symptoms. However, after two weeks p.i., FMDV was only detected routinely in the probang samples. Examination of nasal swabs revealed a higher number of infected animals using RT-nPCR than by the use of the plaque test. Both tests gave approximately equivalent results with the probang samples.

Detection of foot-and-mouth disease virus in nasal swabs of asymptomatic cattle by RT-PCR within 24 hours

A method for extracting RNA from animal-derived materials that provides foot-and-mouth disease viral template suitable for Tth polymerase-dependent synthesis of cDNA and subsequent PCR is described. Viral genomes were detected in less than 24 h. Nasal swabs that can be easily and repeatedly collected, proved suitable for virus detection by PCR, even during the asymptomatic stages of infection.

Indirect foot-and-mouth disease vaccine potency testing based on a serological alternative.

Foot-and-mouth disease (FMD) vaccine potency testing has historically been performed by experimentally infecting vaccinated cattle. A few alternative approaches to the in vivo challenge test based on the correlation between serum titres of primo-vaccinated cattle and protection against infection have been proposed, but none have been accepted by the European Pharmacopoeia (Ph.Eur.) due to the lack of statistical power and the pooling of data over time. The present study addresses these issues and presents data of 150 cattle vaccinated according to Ph.Eur. standards. Four laboratories took part in the serological testing and different serological assays were used, including virus neutralisation assays and ELISA formats. Models correlating specific anti-FMD virus antibody titres to protection were built using logistic regression followed by Receiver Operating Characteristic (ROC) analysis. The best models accurately predicted the in vivo protection status in 80.0% of the cases. Although differences were observed between laboratories and assays used, the majority of antibody pass-levels, determined using ROC analysis, corresponded to at least 75.0% probability of protection. The indirect potency assessment procedure proposed is at least as precise (repeatability=65.8%, reproducibility=60.7%) as the in vivo test, can be standardised and results in a quantitative PD50 value. The validity of the procedure was also demonstrated.

Detection of foot-and-mouth disease virus in the breath of infected cattle using a hand-held device to collect aerosols.

Exhaled air of individual cattle infected experimentally with foot-and-mouth disease virus (FMDV) was sampled to assess the feasibility of a rapid, non-invasive general screening approach for identifying sources of FMDV infection. The air sampler used was a handheld prototype device employing electrostatic particle capture in a microchip chamber of 10-15 μL and was shown to effectively capture a high percentage of airborne microorganisms. The particles were eluted subsequently from the chip chamber and subjected to real-time RT-PCR. Sampling exhaled air for as little as 1 min allowed the detection of FMDV in cattle infected experimentally. Detection in exhaled air from individual cattle was compared to FMDV detection in serum and saliva for 3 different strains of FMDV (O1/Manisa/69, C/Oberbayern/FRG/1960 and SAT1/Zimbawe/1989). Detection of FMDV in exhaled air was possible for all strains of FMDV used for experimental infection but the period that detection was possible varied among the strains. Detection in exhaled air generally peaked on day 2-4 post infection. The perspectives of monitoring for FMDV in the breath of infected cattle are discussed in the context of real-time epidemiological contingencies.

VP1-coding sequences of recent isolates of foot-and-mouth disease virus types A, O and Asia1

A large part of the capsid protein VP1-coding sequence of foot-and-mouth disease virus, isolated between 1993 and 1996 in Europe, was amplified by the reverse transcription-dependent polymerase chain reaction (RT-PCR). The same was done with some non-European virus isolates, especially those against which vaccines were currently produced. The products were sequenced, and the sequences aligned. The alignment comprizes sequences of the types A, O and Asia1. Although the provenance of virus introduced to Europe remains unknown, genetic relation to some other isolates was indicated. Several genotypes of the virus were found to circulate in the field since years.

Characteristics of serology-based vaccine potency models for foot-and-mouth disease virus.

BACKGROUND Foot-and-mouth disease (FMD) vaccine potency testing involves hundreds of animals each year. Despite considerable efforts during the past decades, a challenge-free alternative vaccine potency test to replace the European protective dose 50% test (PD(50)) has not been implemented yet. The aim of the present study was to further characterize the properties of serological vaccine potency models. METHODS Logistic regression models were built for 5 serological assays from 3 different laboratories. The serum samples originated from 5 repeated PD(50) vaccine potency trials with a highly potent A/IRN/11/96 vaccine. Receiver Operating Characteristic analysis was used to determine a serological pass mark for predicting in vivo protected animals. Subsequently, an estimated PD(50) was calculated and the serotype dependency of the logistic models was investigated. RESULTS Although differences were observed between the laboratories and the serological assays used, the logistic models accurately predicted the in vivo protection status of the animals in 74-93% of the cases and the antibody pass levels corresponded to 84-97% of protection, depending on the serological assay used. For logistic models that combine different serotypes, the model fit can be increased by inclusion of a serotype factor in the logistic regression function. CONCLUSIONS The in vitro estimated PD(50) method may be at least as precise as the in vivo PD(50) test and may accurately predict the PD(50) content of a vaccine. However, the laboratory-effect and the serotype-dependency should be further investigated.

Rapid and simple detection of foot-and-mouth disease virus: Evaluation of a cartridge-based molecular detection system for use in basic laboratories.

Summary Highly contagious transboundary animal diseases such as foot‐and‐mouth disease (FMD) are major threats to the productivity of farm animals. To limit the impact of outbreaks and to take efficient steps towards a timely control and eradication of the disease, rapid and reliable diagnostic systems are of utmost importance. Confirmatory diagnostic assays are typically performed by experienced operators in specialized laboratories, and access to this capability is often limited in the developing countries with the highest disease burden. Advances in molecular technologies allow implementation of modern and reliable techniques for quick and simple pathogen detection either in basic laboratories or even at the pen‐side. Here, we report on a study to evaluate a fully automated cartridge‐based real‐time RT‐PCR diagnostic system (Enigma MiniLab®) for the detection of FMD virus (FMDV). The modular system integrates both nucleic acid extraction and downstream real‐time RT‐PCR (rRT‐PCR). The analytical sensitivity of this assay was determined using serially diluted culture grown FMDV, and the performance of the assay was evaluated using a selected range of FMDV positive and negative clinical samples of bovine, porcine and ovine origin. The robustness of the assay was evaluated in an international inter‐laboratory proficiency test and by deployment into an African laboratory. It was demonstrated that the system is easy to use and can detect FMDV with high sensitivity and specificity, roughly on par with standard laboratory methods. This cartridge‐based automated real‐time RT‐PCR system for the detection of FMDV represents a reliable and easy to use diagnostic tool for the early and rapid disease detection of acutely infected animals even in remote areas. This type of system could be easily deployed for routine surveillance within endemic regions such as Africa or could alternatively be used in the developed world.