New and Emerging Technologies: Improved Laboratory and on-site Detection of OIE List a Viruses in Animals and Animal Products
This project is improving the diagnosis of nine major transboundary animal diseases (TADs) - foot-and-mouth disease, swine vesicular disease, vesicular stomatitis, classical swine fever, African swine fever, bluetongue, African horse sickness, Newcastle disease and highly pathogenic avian influenza - by addressing the recommendations of the Scientific Committee on Animal Health and Animal Welfare (SCAHAW). Regarding its epidemiological importance, swine influenza is also included in the project. The programme is focusing on new and emerging technologies, specifically the development, validation and dissemination of robust, specific and sensitive diagnostic tests.
The project is combining the development of improved 'front line' diagnostics, such as dipstick tests that can be used by veterinarians in the field with development of robust and simple nucleic acid and antigen-antibody detection methodologies to be applied in local laboratories and in abattoirs. In addition, the most recent approaches in real-time PCR and in microarray technologies are applied for the improved diagnosis of the nine TADs, notifiable to OIE.[+] Read More
TADs are regularly emerging and re-emerging, threatening animal and human health worldwide (www.oie.int/). To combat these diseases more effectively, the development of rapid, highly sensitive and specific novel diagnostic methods is necessary, including simple and powerful on site tests.
The aim is the complex diagnosis of nine TADs notifiable to OIE. The project is focusing on the development, validation and dissemination of robust, specific and sensitive diagnostic tests, including 'front line' diagnostics, such as dipstick tests.
A real-time RT-PCR assay utilising light upon extension fluorogenic primers (LUX RT-PCR) was developed for the rapid and efficient detection of AIV (Kiss et al., 2006). The LUX RT-PCR technology provided a highly specific and sensitive novel means of AIV detection. Besides its simplicity (use of only two oligos), the LUX technology efficiently reduces the likelihood of false positive results by the use of melting point analysis upon amplification.
A duplex reverse transcription-polymerase chain reaction (dRT-PCR) assay has been developed for the simultaneous, rapid and specific detection of AIV and Newcastle disease virus (NDV) in clinical specimens (Farkas et al., in press). The assay is robust in a sense that it is capable of detecting a broad range of AIV and NDV variants in the same reaction. The speed, simplicity and complexity of the assay facilitate the immediate and front line diagnosis of AI and ND close to outbreak cases.
The assay was shown to be capable of detecting either virus in faecal samples. That makes it an appealing tool among the diagnostic procedures used in monitoring programmes. Therefore, the described dRT-PCR provides a useful and practical supportive method for the effective diagnosis of avian influenza and Newcastle disease.
In order to produce an internal control for the AIV PCR, a 'mimic' has been constructed, which is assuring the reliability of the diagnosis. Rapid one-step real-time PCR (RT-PCR) for the detection (pathotyping and sub-typing) of HPAIV using SYBR Green and TaqMan chemistries is under development.
Two monoclonal antibodies (MAbs 5F10, and HB-65) were evaluated in dipstick tests. Devices for lateral chromatography were prepared using either of the MAbs and specificity testing was carried out. The obtained results show that only MAb 5F10 gave a positive result using the HPAIV strains while MAb HB-65 gave failed to react. MAb 5F10 was able to detect all three HPAIV strains and the negative controls gave a negative result in the experiment.
The objective is the development of ELISAs to identify H5 and H7 viruses using monoclonal antibodies specific for the H5 and H7 haemagglutinin antigens, respectively. Two MAbs out of 42 generated hybridomas specific to H7 and 20 specific to H5 were selected. Sandwich ELISAs were designed for H5 and H7 antigen typing, using a unique MAb, coated to microplate wells as antigen capture antibody, and conjugated with peroxidase as tracer. MAbs 5D8 (H5-specific) and 7A4 (H7-specific) were used.
Analytical sensitivity. Allantoic fluids containing each of four H5 and three H7 subtypes were examined. The detection limit of H5- and H7-ELISA corresponded respectively to 104,5/105 EID50/0,1ml and 104/104.5 EID50/0,1ml.
Analytical specificity. To evaluate the analytical specificity the number of AIVs mentioned above was tested in both H5 and H7 ELISAs. All the H5 subtypes exhibited strong reactivity in the H5-ELISA, in contrast with any other AIV that reacted negative. Similarly, all H7-subtypes, including the equine strain Praga 1/56 H7N7, were positive in the H7-ELISA, in contrast with subtypes displaying other H antigens.
In conclusion, the H5 and H7 ELISAs developed showed a very high specificity, conferred by the two MAbs selected, combined with a broad intra-subtype reactivity that enabled the identification of all the H5 and H7 AIVs examined. Concerning analytical sensitivity, a significant concentration of antigens is needed to produce a positive signal, as expected for antigen detection ELISAs. Then, H5 and H7 typing ELISAs are suited to identify viruses isolated in SPF chicken embryonated eggs and in clinical specimens during acute infection.
Monoclonal antibody-based ELISAs for the detection of antibodies elicited to H5 and H7 influenza viruses were developed as well. Total 813 serum samples were tested by HI assays using the H5 and H7 subtypes of AI strains as antigens. The 5D8 (H5) and 7A4 (H7) MAbs selected for antibody-detection ELISAs have HI activity and recognise an epitope present on all strains tested with the respective H subtype.
H5 and H7 competitive ELISAs. Sensitivity and specificity of the two ELISAs were evaluated using haemagglutination inhibition (HI) test as reference test on both natural and experimental positive sera. For specificity analyses, both naive samples (n=220) and sera originating from animals infected with heterologous subtypes of AIVs were tested. All 173 sera positive in H5-HI scored positive also in H5-ELISA whilst 2 out of 417 sera positive in H7-HI were not detected by H7-ELISA. Two missed sera samples from vaccinated turkeys showed a threshold titre in HI test. Concerning specificity, all 396 sera negative in H7-HI were negative also in H7-ELISA, including 176 sera strongly positive to AIVs of subtypes different than H7.
In conclusion, the correlation between the competitive ELISA and the HI test for the detection of antibodies specific to H5 and H7 AIVs was strongly significant, so that sensitivity and specificity performances of the ELISA evaluated with respect to HI test are very high.
ELISA assays for the detection of antigens and/or antibodies of AIV with main focus on serotypes H5 and H7 described above were developed and improved during the first and second year of the project. At present, they represent rapid, robust, simple tests, which can be used as first line diagnostic test procedures and also in laboratories not equipped with more sophisticated instruments. Improvement of assays in terms of standardisation and diagnostic application was performed and specific monoclonal antibodies are now available.
The developed new diagnostic methods will be disseminated to partner institutes and animal health authorities both in and outside the EU. One of the most important issues in the control of OIE-listed diseases in countries outside the EU is the availability of very simple, easy to use and inexpensive tests like the dipstick tests or diagnostic tests based on portable real-time PCR instruments to be developed in this project. Field veterinarians, simply equipped laboratories and abattoirs can easily use such methods. Simple and effective methods will be provided for the 'first line' diagnosticians.