Recipient Organization
IOWA STATE UNIVERSITY
S. AND 16TH ELWOOD
AMES,IA 50011
Performing Department
VETERINARY MEDICINE
Non Technical Summary
Enteritis in turkeys is an important disease to the US turkey industry due to the economic losses caused by the disease. Multiple agents including several viruses (avian astroviruses, avian enteroviruses, turkey coronaviruses, avian rotavirus (Groups A, D, F, and G), avian reoviruses and turkey toroviruses) are known to cause the disease. (These viruses can be present in different combinations but produce a similar clinical picture making the diagnosis based on symptoms and lesions nonspecific. Control of the disease largely depends on accurate and timely diagnosis of the causative agent/s involved. Current diagnosis is done by electron microscopy (EM), immunoelectron microscopy (IEM), immunofluorescent antibody technique (IFT) and enzyme-linked immunosorbent assay (ELISA). Availability of a robust diagnostic test for rapid and simultaneous detection of viral causes of turkey enteritis will be immensely useful for implementing effective control strategies and thus
reducing the economic losses to the industry caused by this disease. Additionally, rapid and accurate diagnosis of viral causes will prevent indiscriminate use of antibacterial agents and costs associated with ineffective therapeutic interventions. The main aim of the proposed research is to develop, harmonize and standardize a multiplex, reverse transcription PCR (RT-PCR) technique for robust, rapid and accurate detection of six viruses (avian reoviruses, avian astroviruses, avian enteroviruses, turkey coronaviruses, avian rotaviruses and turkey toroviruses) that are known to cause enteric disease in turkeys. We will use the available genomic databases of poultry enteric viruses as well as published literature to select the primers and probes to develop the test. Sample enrichment and nucleic acid extraction procedures, and internal controls will be developed to increase the sensitivity of the assay and to minimize the contamination risk. First, the assay will be optimized, evaluated
in our laboratory, and passed through the internal quality assessment procedures. Later, it will be subjected to external quality control assays with the participation of other Veterinary Diagnostic Laboratories in the country before disseminating the test procedure.
Animal Health Component
50%
Research Effort Categories
Basic
(N/A)
Applied
50%
Developmental
50%
Goals / Objectives
Overall objectives of this project are to develop, harmonize and standardize a multiplex PCR technique for rapid and accurate detection of six viruses (avian reoviruses, avian astroviruses, avian enteroviruses, turkey coronaviruses and turkey toroviruses) that are known to cause enteric disease in turkey. To accomplish this goal and to test our hypothesis we will 1) develop a mutiplex RT-PCR for direct and simultaneous detection of avian reoviruses, avian astroviruses, avian enteroviruses, turkey coronaviruses, avian rotaviruses, and turkey toroviruses from turkey intestinal samples, dropping samples and/or cloacal swabs; 2) produce a library of internal quality controls to use in the developed technique; and 3) subject the multiplex RT-PCR to internal and external quality assurance of the test procedure
Project Methods
To test our hypothesis, state-of-the art molecular approaches are proposed. Overall we plan to develop a multiplex PCR for simultaneous detection of viruses causing enteritis in turkeys from cloacal swabs. Specific objective 1a. To develop a multiplex real time for direct simultaneous detection of avian reoviruses, avian astroviruses, avian enteroviruses, turkey corona viruses, avian rotaviruses, and turkey toroviruses from turkey intestinal samples/dropping samples/cloacal swabs. First, we will attempt to use the TaqMan real time PCR assay. In the event that this procedure fails, we will attempt other real time PCR formats, such as molecular beacons. Since avian rotaviruses include 4 serogroups, the initial multiplex PCR may be limited to detecting genetic material indicating the presence of a rotavirus. If this proves to be the case, then it may be necessary to develop a separate rotavirus group panel to identify different serogroups. Specific objective 1b Convert
Real-time PCR format to Luminex Bead format. Because currently available real-time PCR instrumentation limits detection to 4 or 5 different fluorescence channels, we will attempt to convert the real-time assays to Luminex bead technology. This method allows for detection of up to 100 targets per reaction. The probes will be resynthesized but instead of incorporating the normal TaqMan fluorescent dyes, the oligonucleotides will be coupled with Luminex beads. With this technology, standard multiplex PCR is performed, followed by hybridization with the luminex-bead labeled probes. The final product is loaded into an instrument (BioRad Bio-Plex) where signal is detected for any and all of the targets that are present. This technology has been successfully used for a number of human and veterinary pathogens. If we are unsuccessful with this approach, we will instead develop two smaller (3 agents each) standard real-time multiplex assays to incorporate all of the agents of interest.
Specific objective #2. To produce a library of internal quality controls to be used in the PCR. One concern with RT-PCR is the potential for false-negative results due to the presence of RT-PCR inhibitors, poor target RNA recovery during extraction, degradation of target RNA before amplification, errors in setting up a reaction, or a degraded reagent. Therefore, the presence of an internal positive control (IPC) in each sample would be useful to monitor the RT-PCR procedure and to ensure that the test was performed correctly, thereby giving an acceptable level of confidence for the absence of false-negative results. We will use the protocol by Tang et al. (2005) to prepare single stranded RNA (ssRNA) IPC template reagents. These ssRNA fragments will be designed so that they can be monitored in the same reaction using different fluorescent probes in a multiplex RT-PCR. Once all the steps of the RT-PCR assay are optimized, we will prepare a standard optimization protocol for each step
of the test procedure. Our plan is to validate the test results with the participation of 5 selected Veterinary Diagnostic Laboratories in the United States.