Progress 07/01/23 to 06/30/24
Outputs
Target Audience:Conduct in-person and zoom meetings with veterinarians specialized in the turkey industry to discuss our project and to explain the significance of non-invasive sampling as a novel method for early detection of turkey reoviruses. This personalized approach allows for in-depth discussions and addressing specific concerns. Conduct in-person workshops for veterinarians to train them on proper collection and preservation techniques of non-invasive samples from commercial turkey farms. Additionally, it included the guidelines for the proper shipment of samples to our lab. This interactive approach ensures proper sample collection, handling, preservation, and shipment for accurate testing. North Central Avian Disease Conference (NCADC) presentation: we presented our research effectively at the NCADC. This presentation helped to reach a broad audience of industry professionals. The audience was a collection of poultry veterinarians, turkey-producing companies, professors, and experts in turkey production and diseases. Interestingly, it also included several discussions with veterinarians and producers interested in recruiting their farms for our project. In-person training was conducted for virology section at the Animal Disease Research and Diagnostic Laboratory (ADRDL) to train them for sample processing (tendons, tissues, and non-invasive samples) for virus isolation and real-time RT-PCR. Changes/Problems:Due to continuous outbreaks of highly pathogenic avian influenza viruses, we are not able to visit different farms for samples collection and on farm training. We are using online tools to educate producers and field veterinarians. We are also scheduling in-person meetings with field veterinarians to train them for sample collection and to provide them with sampling kits. We are also sending sampling kits through courier services. What opportunities for training and professional development has the project provided?We conducted a series of online and in-person workshops specifically designed for veterinarians working in turkey production. We trained veterinarians to collect environmental samples from turkey farms. This training emphasized the benefit of early detection, in implementing proactive measurements before clinical signs appear in the flock and mitigate the devastating impact of the infection. They were able to understand the importance of collecting non-invasive samples during the pre-placement period (after cleaning and disinfection but before poults arrival). This step is very helpful for veterinarians to evaluate the effectiveness of cleaning and disinfection programs as well as to decide on repeating cleaning and disinfection if needed to ensure complete eradication of TRV before restocking. Additionally, importance of testing environmental samples from commercial turkey hatcheries was highlighted as a method to ensure that the one-day-old poults that will be received by the commercial turkey farms are free from TRV. This method will help the producers to receive TRV-free poults that will reduce the incidence of infection and significant economic losses. The Participants gained valuable knowledge on collecting representative samples from various sites in the farm environments. Veterinarians were educated with the required skills to label and store samples for precise identification, as well as preserve and ship the samples properly to the lab. Two post-doctoral associates at the UMN and one post-doctoral associate at the SDSU were mentored for professional development. One graduate student is also being involved at the SDSU to learn about turkey reoviruses. All (post-docs and student) were able to understand diseases caused by different variants of turkey reoviruses. They were able to understand turkey production system and challenges associated with control and prevention of disease cause by turkey reoviruses. How have the results been disseminated to communities of interest?We have actively disseminated the exciting findings of our research to a wide range of stakeholders in the turkey industry. We conducted online and in-person meetings specifically for poultry veterinarians to discuss and implementing our findings. Results were presented at the North Central Avian Disease Conference (NCADC). We will also present our findings at the American Association of Avian Pathologists (AAAP) annual conference to be held in July 2024, reaching to a broad international scientific community. All our findings and results will be published in scientific journals to ensure wide accessibility. What do you plan to do during the next reporting period to accomplish the goals?Goal One: Develop nomenclature and a genotypic classification method for turkey reoviruses We will continue to work on defining nomenclature and genotyping classification. New sequences from environmental samples will be included. Training materials will be developed for workshops mainly for field veterinarians, graduate students, molecular diagnosticians at veterinary diagnostic laboratories and researchers for better understanding of genotyping classification and interpretation of whole genome sequence analysis. Goal 2: Validate real-time RT-PCR (RT-qPCR) for environment samples Continue to evaluate diagnostic sensitivity of this assay mainly for environmental samples as well as for detection of emerging variants of TRVs. Goal 3: Develop a non-invasive sampling method to detect turkey reoviruses We will continue to screen more farms with a known history of TRV infection and clean farms with no TRV infection reported in the past. Goal 4: Conduct whole genome sequencing and genotyping of selected biosurveillance samples Biosurveillance samples for virus isolation and real-time RT-PCR. The selected positive samples will be processed for whole genome sequencing. Goal 5: Evaluate various decontamination technologies Experiments are in progress to test the other two strains for evaluation of inactivation of turkey reovirus strains by a commercial device using ultraviolet light and ozone h. All six TRV isolates will be tested with H2O2-producing technology and misting with AquaPure AquaLyte. The focus will be on specific aim B (extension) to prepare educational materials related to all goals.
Impacts
What was accomplished under these goals?
Goal One: Develop nomenclature and a genotypic classification method for turkey reoviruses (25% Accomplished) The South Dakota State University and University of Minnesota teams are working on defining nomenclature and genotyping classification. We are working on (n = 257) TRV whole genome sequences grouped into seven geographic regions: Canada; Dakotas (ND, SD); Midwest (KS, MO, AR); Upper Midwest (IA, WI); Minnesota (MN); North Central (MI, IN, OH, KY); and East (PA, VA, NC, SC). This grouping was based on turkey density, state adjacency, and sequence availability. Bayesian phylodynamic analyses were performed using the final sequence alignment of all ten TRV segments as input to estimate the evolutionary dynamics and spreading patterns of the virus. This extensive data analysis will make a base for proposing nomenclature and genotyping classification. Goal 2: Validate real-time RT-PCR (RT-qPCR) for environment samples (80% Accomplished) Our research team has successfully validated a universal real-time RT-PCR assay for detecting avian reoviruses from environment samples. This assay demonstrated high sensitivity and specificity in the detection of TRV in the environment (non-invasive) samples. Importantly, the results confirmed the absence of any inhibitory substances within these samples that could interfere with the samples processing, nucleic acid extraction and real-time RT-PCR. This high sensitivity of the assay is extremely important for detection of TRV even if virus is present in low concentration. Goal 3: Develop a non-invasive sampling method to detect turkey reoviruses (80% Accomplished) We developed a cost-effective in-house sampling kit for early detection of turkey reovirus. These kits are sterile cotton gauze pre-soaked in brain heart infusion broth with antibiotics and antifungals (to prevent contamination) and were shipped to the recruited farms at 4°C. Farm staff used the kits to collect environmental samples weekly, starting before poults arrival (pre-placement) until they were marketed. Samples were typically collected from surfaces like walls, drinkers, fans, and litter. After collection, the samples were shipped back to our lab at 4°C and stored at -80°C until processing. This method allowed us to detect turkey reovirus RNA in the farm environment before any clinical signs of infection appeared in the turkeys. We also succeeded in the isolation of live turkey reoviruses from the farm environment from foot covers, fans, and walls which was the report of virus isolation from the environment. Furthermore, the application of a noninvasive sampling strategy at the preplacement time helped in the assessment of the cleaning and disinfection programs used in the farms. Interestingly, in the preplacement sampling, we detected the turkey reovirus RNA and infectious virus in noninvasive samples collected from the walls, indicating that cleaning and disinfection efforts were insufficient to eradicate the virus. This early detection allows for corrective actions to be taken before the turkeys arrive, minimizing the risk of infection. Hence, there is no doubt that this approach will be very helpful in evaluating cleaning and disinfecting strategies at turkey farms. This effective cleaning and disinfecting will be helpful in reducing TRV infection in new flocks. Goal 4: Conduct whole genome sequencing and genotyping of selected biosurveillance samples (20% Accomplished) As mentioned in goal 3, we have isolated TRV from environmental samples. We are planning to do whole genome sequencing. Goal 5: Evaluate various decontamination technologies (30% Accomplished) The evaluation of inactivation of four turkey reovirus strains (TERV, TARV-1, TARV-O'Neil, TBRV) by a commercial device using ultraviolet light and ozone has been completed. Experiments are in progress to test the other two strains. A commercial device called the "PathO3Gen Solutions UVZone Shoe Sanitizing Station (SSS)" was obtained from Pathogen Solutions (St. Petersburg, FL). This machine produces ozone (6.0 ppm) at a wavelength of 185 nm and UV-C (2000 lW/cm2 ) at a wavelength of 254 nm. The machine has four UV (386 mm) and two ozone (357 mm) lamps and is certified by a Nationally Recognized Test Laboratory for safe use. The machine sterilization cycle, which lasts a total of 8 sec, begins automatically when someone stands on the glass shield of the machine. All experiments were conducted at room temperature (25º C) with 50% relative humidity. Six different fomites were tested, namely aluminum (AL), rubber boot (RB), cardboard (CB), denim fabric (FB), polypropylene (PP), and stainless steel (SS). The surviving viruses were eluted from all fomites using 100 µl of elution buffer (3% beef extract in 0.05 M glycine) per fomite. Serial 10-fold dilutions of all eluates were prepared in maintenance medium for virus titration. The experiment was repeated once, and the amount of average virus reduction was calculated by subtracting virus titer in "treated" fomites from those in "control" fomites In general, the SSS shoe sterilization machine was able to kill more viruses on porous fomites than on non-porous ones. No significant difference was observed in the inactivation rate among the four different reovirus strains. Hence, disinfection strategy using SSS shoe sterilization machine should be effective irrespective of reovirus type.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2024
Citation:
M., Selim, Jangra, S, Luqman, M., Temeeyasen, G., Ohnstad., M, Long., C, Sharafeldin, T., Mor, S. Validation of non-invasive sampling as a diagnostic tool for early detection and screening of avian reovirus in turkey flocks. The 75th North Central Avian Disease Conference (NCADC) was held in Minnesota on the 16th and 17th of April.
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