Menu
Development of a serologic assay and novel vaccine approach to mitigate turkey arthritis reovirus
Requested fund from MTRPC: $70,000
Project duration
From 9/1/2021to 8/30/2023
Principal Investigator
Tamer Sharafeldin, DVM, MVSc, PhD
Assistant Professor (Veterinary Pathologist), South Dakota State University Animal Disease Research and Diagnostic Laboratory
N campus Dr, Brookings, SD, 57007Tamer.Sharafeldin@sdstate.edu Office: 605/688-6544
Main objectives
The main objectives are to improve the diagnostics capability and the vaccination strategy to limit the economic losses induced by turkey arthritis reovirus. To fulfill these objectives, we outlined 3 specific aims to be completed through clear and measurable laboratory and experimental procedures as follows:
1. Develop a sensitive and specific Indirect Fluorescent Antibody (IFA) assay to detect turkey reovirus antibodies.
2. Develop and evaluate turkey reovirus-ISCOMs vaccines.
3. In vitro and in vivo evaluation of BacMam vector carrying turkey reovirus genes.
Develop a sensitive and specific Indirect Fluorescent Antibody (IFA) assay to detect turkey reovirus antibodies: After thorough discussion among the research team considering the logistics of diagnostics, we decided to develop ELISA instead of IFA.
TARV-SigmaC (S1 gene) sequence was codon optimized and cloned into pET-28a plasmid then transformed into competent DE3 E. coli. The expression of target gene was verified by western blot and once verified, the expressed protein was purified and concentrated to be used to coat plates for ELISA. Standardization and optimization of Turkey reovirus ELISA assay was performed to determine of the appropriate antigen concentration, blocking buffer, blocking Ime, sample diluent and the conjugated anti-turkey HRP. During the standardization, sera from known positive and known negative are used. We are currently validating the assay by testing field serum samples, and we establish relative sensitivity and specificity. The result of our developed assay is compared to the results of the commercially available chicken reovirus ELISA kit. So far, we received nearly500 serum samples with the help of turkey veterinarians in Minnesota, South Dakota, North Carolina, and other states. We expect to have nearly
1000 serum samples for validation. So far, the assay is showing very promising results, and we expect to deploy it in fall 2023.
Develop and evaluate turkey reovirus-ISCOMs vaccines: The ISCOMATRIX was developed in our laboratory by using Quil A:Cholesterol: Phospholipid in ratio of 5:1:1.This suspension was dialyzed, and filtrated. The suspension was visualized the negative staining electron microscopy to verify the assembly of cage like structure particles(Fig.1). The purified, and concentrated TARV-SC antigen, which was produced in E. coli expression system, was mixed with the ISCOMATRIX suspension. The ISCOM-TARV-SC integration in the mix was verified using immunogold electron microscopy (Fig2). The protein was quantified, and the final protein concentration is0.5 mg/ ml. The vaccine is ready to be administrated to SPF turkeys to test the efficacy and safety (Trial start on September 16/date of receiving the SPF turkeys).
Fig1: ISCOMATRIX particles of nearly 35nm of diameter.
Fig2: ISCOMATRIX particles (Icosahedral) conjugated with the SigmaC protein (spiral shape)
In vitro and in vivo evaluation of BacMam vector carrying turkey reovirus genes: Sequences of TARV- sigma C & B were codon optimize and cloned to baculovirus transfer vector. Both the cloned vector and the linearized baculovirus DNA were transfected to sf-9 cells. The success of gene transfer from the shuttle vector to the backbone of baculovirusis confirmed through the visualization under fluorescent microscopy. Once the virus assembly is confirmed by the positive green fluorescent signal. The virus was propagated on sf-9 cells and the expression of the cloned genes were confirmed by western blot. Once the western blot confirmed the expression of both TARV-sigmaC and SigmaB, both recombinant viruses were propagated and titrated. The r-baculoviruses were mixed 1:1 before vaccine administration. The first trial showed that birds vaccinated with 2 doses of the vaccine via oral/intranasal routes at 2 and 4 weeks had a significantly lower tendon lesion scores after challenge at 5 weeks of age compared with the non-vaccinated control(Fig3). A second trial to confirm safety and efficacy is to start on September 16 upon the arrival of the SPF turkeys.
Fig3: The histologic lesions score of turkeys which received BAcMam-SigmaC-SigmaB vaccine vs controls. The higher lesions scores reflect more disease and less protection. Turkeys received vaccines had significantly lower tendon histologic lesion scores after challenge compared with challenge control group which did not receive the vaccine. The group that received 2 doses of the BacMam-SigmaC-SigmaB vaccine had no significant difference after challenge in comparison with the negative control group which was not challenged.
Final comment: The ELISA assay is developed, and validation process is currently performed. The assay will be offered for diagnostics this fall. Both vaccines were developed and there combinant baculovirus showed a promising result. SPF turkeys delayed submission created some delay. We expect submitting the final report by the end of fall 2023.
