Due to the similarities between different viruses in Coronavirus family, it is difficult to differentiate between them. Therefore, a proper laboratory diagnosis is important to characterize PEDV. Broadly, we classify diagnostic methods into Virological assays and serological assays.
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Vero cells are the cell lines that are suitable for the propagation of PEDV. Immunofluorescence Assay (IFA) and RT-PCR are the best techniques for PEDV isolation from cell culture. Other cell lines like the Primary intestinal epithelial cell or any other cells that express porcine aminopeptidase N (APN), can help to propagate PEDV in cell line. Immunohistochemistry (IHC) is a technique for the detection of PEDV. They use antibody-antigen and enzyme-substrate reactions. Through IHC, PEDV can be visualized on target cells. IHC has another advantage too because the samples which we prepared during IHC long-term has long-term storage.
Immunofluorescence (IF) assay helps in the identification of PEDV antigens infecting the cells. IFA detects viral antigens (antibody-antigen reaction) in cell culture. Intra- fluorophore-conjugated PEDV-specific antibodies are used to detect viral antigens. Under a fluorescent microscope, viral antigens, are visualized in the cytoplasm of infected cells during various phases of infection. ELISA detects virus antigen using PEDV-specific antibodies. Firstly, the plate is coated with the PEDV-specific capture antibody overnight and incubated with the sample. The antibody-antigen binding, and the reaction is developed by adding an enzyme-linked secondary antibody . Limitations may be how and when the samples were collected and stored. Real-time Reverse transcriptase-polymerase chain reaction (r RT-PCR) assays are methods of choice for the diagnosis of PEDV. PEDV genome in feces, rectal swabs or intestinal samples from animals can be used as a sample. PCR detects PEDV genes based on one of the structural proteins. In conventional RT-PCR, the virus-specific primers target the PEDV genome and amplify it to the thousands of copies. The process is then followed by electrophoresis in agarose gels. RT-PCR detects genes by using non-specific fluorescent dyes for double-stranded DNA. The major advantages of RT-PCR assays are that they are highly sensitive and able to detect the target sequences simultaneously with the amplification reaction.
Serological examination measures the level of exposure of the animal herd to the virus. Detection of PEDV specific antibodies helps to know about the sow immunity and to track the neonatal immunity level. The principle is based on antibody-antigen reaction where PEDV infected Vero cell cultures are used as antigen and antibodies are already present in serum samples from suspect animals. The antibodies are detected with fluorophore-labeled anti-porcine secondary antibodies and visualized under a fluorescence microscope. The main advantage of IFA is that it is less time consuming and easier to perform.
Virus neutralization assays (VNs) are also one of the serological assays that are widely used for detection of PEDV. It is the process to check viral infectivity. The neutralizing antibodies bind to the virus(antigens) and block one or more steps of the viral. Formation of CPE is used to detect neutralizing antibodies against PEDV. In indirect ELISA, the viral antigen is coated in the plate and the test sample is added to the plate. Antibody-antigen complex is formed when the sample is incubated using an enzyme-linked secondary antibody. Competitive ELISAs works on the principle that when the antigen is added to microplates coated with the antibodies, a reaction is developed based on the PEDV-specific monoclonal or polyclonal antibodies. The blocking ELISA is more specific when compared to the indirect ELISA.
Proper disinfection and biosecurity are the most important measures to prevent acute PED outbreaks. PEDV can survive in feces, feed, and water for extended periods. PEDV can survive in fresh feces up to 1 week at 40oC, dry feed for 1 week at 25oC, wet feed for 4 weeks at 25oC, drinking and recycled water for 1 week at 25oC and slurry for 2 weeks at 25 ͦ C. The proper and strict application of disinfection blocks the entry of pathogens to the farm. Disinfection must be thoroughly applied to all fomites, personnel, and external visitors that could be contaminated with PEDV. PEDV is inactivated by most viricidal disinfectants but sometimes PEDV RNA can be detected by RT-PCR even though disinfection is done. Firstly, cleaning should be done under high pressure and water at high temperature. Secondly, disinfection by an appropriate disinfectant and thirdly, drying it overnight. The crowd people should be restricted between fattening and farrowing such that there will be less contact between trailers and the farm interior New animals should be quarantined before they are shifted to the farm.
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