PATHOGENESIS AND CONTROL OF EMERGING REOVIRAL ARTHRITIS IN TURKEYS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: ACTIVE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1025754
• Grant No.: 2021-67015-34465
• Cumulative Award Amt.: $500,000.00
• Proposal No.: 2020-06619
• Project Start Date: Jul 1, 2021
• Project End Date: Jun 30, 2026
• Grant Year: 2021
• Program Code: [A1221]- Animal Health and Production and Animal Products: Animal Health and Disease

Recipient Organization
OHIO STATE UNIVERSITY
1680 MADISON AVENUE
WOOSTER,OH 44691

Performing Department
OARDC Food Animal Health

Non Technical Summary
Turkey reoviruses causing lameness in market age turkeys have been recognized in the US since 2011. Significant economic losses are incurred due to poor weight gain, mortality, and condemnations at the processing plants. Currently, there is no effective measures to control and prevent reoviral arthritis in turkeys. To lay the groundwork for development of effective control measure, emerging turkey reoviral arthritis needs to be comprehensively defined. In this study, we will study turkey reoviral arthritis in context of age-based host responses. We will evaluate the effect of age on tissue tropism of the virus, level of virus replication, and the onset and severity of the disease. We will also investigate how natural infection-induced and passively acquired maternal immune responses affect the onset and development of reoviral arthritis. In addition, we will determine how microbiome modulation by probiotic affects disease outbreak and severity. Our study will not only enhance our understanding of the mechanism of turkey arthritis but also have the potential of unraveling a novel method of controlling reoviral disease in turkeys. Additionally, information obtained from this study can be applied to develop control and prevention measures against other economically important poultry enteric viral infections.

Animal Health Component

25%

Research Effort Categories

Basic

75%

Applied

25%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3230	1090	20%
311	3230	1100	20%
311	3230	1101	50%
311	3230	1160	10%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3230 - Turkey, live animal;

Field Of Science
1090 - Immunology; 1101 - Virology; 1160 - Pathology; 1100 - Bacteriology;

Keywords

reovirus

arthritis

microbiome

vaccine

Goals / Objectives
Turkey arthritis reoviruses (TARVs) have been recognized in the US since 2011 to cause disease in market age turkeys. Novel strains continue to rise from frequent reassortment of TARVs with other field strains and rapid spontaneous mutations attributed to viral RNA-dependent RNA polymerase errors. Significant economic losses are incurred due to poor weight gain, increased feed conversion ratios, mortality as a result of culling or aortic rupture, and condemnations at the processing plants. Control measures currently being employed by some commercial farms are not effective mainly because the disease is not well understood. Attempts to understand TARV pathogenesis are complicated by the high prevalence of apparently "nonpathogenic" reoviruses in the intestines of commercial turkeys. The high prevalence of reoviruses in commercial settings throughout production cycles has also made it difficult to determine the age at which turkeys are susceptible to TARVs. Further, our field and experimental observations in commercial and specific-pathogen free (SPF) turkeys suggest that gut microbiota plays a role in initiation and establishment of TARV infection and subsequent induction of arthritis.Turkey reoviral arthritis needs to be comprehensively defined to lay the groundwork for development of effective control measures. The goal of the proposed work is to understand how viral, host, and microbial factors contribute toward the onset, development, and severity of reovirus-associated arthritis in turkeys. This goal will be accomplished through the following objectives: Objective 1. Define turkey reoviral arthritis in context of age-based host responses. We will evaluate the effect of age on tissue tropism of the virus, level of virus replication, and the onset and severity of the disease. We will also investigate how natural infection-induced and passively acquired maternal immune responses affect the onset and development of reoviral arthritis. Objective 2. Determine how microbiome modulation affects disease outbreak and severity. Turkey-specific microbiome modulator (probiotic) will be applied in a multi-omics approach to investigate how the disease is influenced by the interaction between microbiome, TARV, and the host.

Project Methods
We will evaluate the effect of age on tissue tropism of the virus, level of virus replication, and the onset and severity of the disease. Specific-pathogen-free (SPF) and commercial turkeys will be infected with TARV at different weeks of age. The overall effect of TARV on turkey performance phenotype will be determined by tracking the following parameters: body weight gain, weight of feed consumed, morbidity, and mortality. To monitor virus shedding, cloacal swabs will be taken at 3 days and 1-week post infection and continue weekly until there is no detectible virus in cloaca. The birds will also be weighed every week to detect weight gain depression. At 4 week post infection (WPI), 6 poults from each group will be euthanized for sample collection. Samples from tendons (gastrocnemius and digital flexor tendons) will be used for virus detection and quantification. In addition, hock joint and gut samples will be collected and fixed in 10% neutral-buffered formalin for histopathological detection of tenosynovitis (in tendons) or enteritis (in intestines) and typhlitis (in ceca). The remaining birds will be monitored for development of clinical signs till 16 WPI and samples collected as described above.We will investigate how immune responses induced by natural infection or passively transferred to poults through the yolk affect the onset and development of reoviral arthritis. To gain more insight into the mechanism of pre-existing "immunity" and superinfection (or co-infection) with "nonpathogenic" reoviruses, a controlled experiment will be conducted with SPF birds. A group of one day-old SPF poults will be infected with non-pathogenic reovirus. The birds will be challenged one week later with TARV virus and raised and sampled as described above. In another experiment, ten-day-old embryonating SPF turkey eggs will be injected in the yolk sac with 0.2 ml of hyper-immune pools of reovirus anti-sera. Poults will be bled to determine the acquired antibody titers and also challenged with TARV virus at one week of age. The age of infection may be changed depending on data from first experiment. Data collection and euthanasia will be done as described.We will determine how functional modulation of the gut microbiome with turkey-specific probiotics, which positively influences the production performance and the immune system, affects the host's immune responses to TARV infection and the course of turkey reoviral arthritis. In first experiment, turkey-specific probiotic strains will be supplied in crumbled feed ad libitum as dose of 1×108 CFU/strain or species/gram of feed and discontinued at 8 weeks of life, at the peak of microbiome maturity. We will determine how colonization by turkey-specific probiotics and bacterial microbiome shifts induced by the probiotic supplementation relate to the course of turkey reoviral arthritis. In second experiment, we will determine the translatability of data in commercial turkeys. Single- or multi-strain probiotics will be selected or formulated based on an integrated analysis of virologic, pathogenic, production, and microbiome data obtained from first experiment. The main criteria for selection of probiotics will include, but not limited to, the ability of the probiotics to decrease viral load and disease parameters while improving bird performance parameters. Furthermore, we will assess functional modulation of recall immune responses by probiotics supplementation. This will enable correlation analysis between recall immune responses (PBMC transcription modules and stimulation index) in TARV-infected birds, tenosynovitis, and clinical lameness.

Progress 07/01/23 to 06/30/24

Outputs
Target Audience:The poultry industry including backyard poultry farmers; government and state officials involved in the control of animal diseases. Scientists in the fields of infectious diseases, animal production, Ag-engineering, virology, bacteriology, immunology & vaccinology. Extension and outreach specialists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One graduate student and two research assistants are currently working on this project. Graduate student attended and presented scientific research at internal and international conferences. Graduate student attended the Tri-State Poultry Veterinarians Meeting Graduate student has learned appropriate techniques for necropsying turkeys and preparing tissues for immunohistochemistry analysis. Graduate student learned techniques for collecting and analyzing microbiome samples. How have the results been disseminated to communities of interest?Our findings have been presented at the CRWAD (Jan 21-24, 2024) held at Chicago, Illinois; 37th Annual Hayes Advanced Research Forum 2024 (Feb 24, 2024), CFAES Annual Research Day (April 09, 2024), The Ohio State University, Columbus, Ohio; and at the American Society for Virology (ASV) meeting (June 24-28, 2024), Columbus, Ohio. What do you plan to do during the next reporting period to accomplish the goals?The future work will help to determine: 1.Role of pre-existing immunity or concurrent coinfection with ubiquitous reovirus and TARV affects the onset and development of disease 2.Impact of microbiota disruption on turkey performance 3.Role of microbiota in outbreak and severity of reoviral arthritis 4.The role of gut-joint axis in reoviral arthritis 5.Whether probiotics, microbiome-based intervention can work to alleviate reovirus induced arthritis.

Impacts
What was accomplished under these goals? Objective 1. Define turkey reoviral arthritis in context of age-based host responses 1(A): Age related factors within the host: To evaluate the effect of age on the onset and severity of reoviral arthritis in turkeys, an experimental turkey infection study was completed. Commercial turkey poults, derived from breeder hens vaccinated with autogenous vaccines and with known reovirus antibody endpoint titers, were orally inoculated with 4×106 TCID50 of TARV O'Neil at 1, 3, and 7 weeks of age (WOA) and euthanized at 3-, 7-, and 28-days post infection (dpi). Cloacal virus shedding, virus isolation from tendons, and virus migration to tendons was measured. The hock joints were processed to prepare slides and were kindly scored by Dr. Saad, Poultry Pathologist, University of Minnesota following previously described scoring system (Sharafeldin et al., 2015). TARV-induced weight gain suppression was evident in poults infected at one week of age. Age-dependent variations in cloacal viral shedding and inflammation severity in tendons were also observed. Moreover, microbial beta diversity analysis showed dissimilarity in bacterial communities between mock and infected groups. Conclusively, maternally derived antibodies, derived from breeder hens vaccinated with autogenous vaccines, appeared insufficient cloacal viral shedding and subsequent pathological changes in tendons due to challenge virus. 1(B): Effect of naturally induced and passively acquired immunity: To determine how pre-existing immunity or concurrent coinfection with the ubiquitous reovirus and TARV affects the onset and development of disease in SPF turkeys. For the coinfection study, the coinfected group of poults were infected with TERV MN1 virus at 1 day of age and TARV O'Neil at 8 days of age, while O'Neil infected group was only infected with TARV O'Neil at 8 days of age. Results demonstrated the average weight gain is found to be significantly higher in coinfected group when compared to O'Neil only infected groups. The coinfected group showed higher antibody titers at 2 weeks post infection with O'Neil, in comparison with O'Neil infected group. Additionally, the samples are being analyzed for metatranscriptomics, viral tendon tropism and histologic inflammation scores of tendons. To analyze the effect of pre-existing immunity on the onset and development of arthritis due to TARV, the hyper-immune reovirus anti-sera (anti-O'Neil and anti-MN1 serum) was injected into the yolk sac of 10-day-old embryonating SPF turkey eggs. After hatching, the poults were orally challenged with TARV O'Neil at 7 days of age. Samples were harvested at 1- and 4-weeks post infection. The samples are being analyzed for antibody response, viral tendon tropism, tendon histologic lesion, and cloacal shedding. Objective 2: Determine how microbiome modulation affects disease outbreak and severity. The study was conducted to determine the functional modulation of gut microbiome with turkey specific probiotic strains (Lactobacillus aviarius subsp. aviarius, Lactobacillus aviarius subsp. araffinosus) affecting host immune response to TARV infection and course of arthritis. In an initial study, SPF turkeys were orally inoculated with 200 µl of 108 CFU/strain/bird from 0 day of age to 2 weeks of age (14 days), and infected groups were orally inoculated with TARV O'Neil at 1 week of age. The poults were euthanized at 4 weeks post-infection (4 wpi) and samples were collected. The samples are being analyzed for antibody response, viral tendon tropism, tendon histologic lesion, and cloacal shedding.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Saroj Khatiwada, John Ngunjiri, Patricia Boley, Kush Yadav1, Carolyn Lee1, Chang-Won Lee, Saad Gharaibeh, Gireesh Rajashekara, Scott P Kenney.Pathogenesis of reoviral arthritis in turkeys derived from vaccinated breeder hens. CRWAD, Jan 21-24, 2024
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Khatiwada, S., Ngunjiri, J., Boley, P. A., Yadav, K. K., Ghorbani, A., Lee, C. M., Lee, C.-W., Rajashekara, G., & Kenney, S. P. (2024, February 24). Evaluation of reoviral arthritis in commercial turkeys derived from vaccinated breeder hens. 37th Annual Edward F. Hayes Advanced Research Forum.
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Khatiwada, S., Ngunjiri, J., Boley, P. A., Yadav, K. K., Ghorbani, A., Lee, C. M., Lee, C.-W., Gharaibeh, S., Rajashekara, G., & Kenney, S. P. (2024, April 9). Evaluation of reoviral arthritis in commercial turkeys derived from vaccinated breeder hens. CFAES Research Forum.
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Khatiwada, S., Ngunjiri, J., Boley, P. A., Yadav, K. K., Ghorbani, A., Abundo, M., Lee, C. M., Poelstra, J., Lee, C.-W., Gharaibeh, S., Rajashekara, G., & Kenney, S. P. (2024b, June 24). Age dependent susceptibility to turkey arthritis reovirus in presence of maternally derived antibodies. American Society for Virology (ASV).
• Type: Journal Articles Status: Submitted Year Published: 2024 Citation: Khatiwada, S., Ngunjiri, J., Boley, P. A., Yadav, K. K., Ghorbani, A., Abundo, M., Lee, C. M., Poelstra, J., Lee, C.-W., Gharaibeh, S., Rajashekara, G., & Kenney, S. P. (2024a). Age based host response to turkey arthritis reovirus in commercial turkeys in the presence of maternally derived antibodies.

Progress 07/01/22 to 06/30/23

Outputs
Target Audience:The poultry industry including backyard poultry farmers; government and state officials involved in the control of animal diseases. Scientists in the fields of infectious diseases, animal production, Ag-engineering, virology, bacteriology, immunology & vaccinology. Extension and outreach specialists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One graduate student, two research assistant are currently working on this project and one Research Scientist worked on this project past year How have the results been disseminated to communities of interest?The results from this study are being disseminated through conferences, publications and meetings. What do you plan to do during the next reporting period to accomplish the goals?The future work will help to determine: Role of pre-existing immunity or concurrent coinfection with ubiquitous reovirus and TARV affects the onset and development of disease Viral, host, and intrinsic microbial factors contributing toward the onset, development, and severity of reovirus-associated arthritis in turkeys Host/viral factors responsible for microbiota disruption Impact of microbiota disruption on turkey performance Role of microbiota in outbreak and severity of reoviral arthritis The role of gut-joint axis in reoviral arthritis Whether probiotic microbiome-based intervention can work to alleviate reovirus induced arthritis.

Impacts
What was accomplished under these goals? Objective 1. Define turkey reoviral arthritis in context of age-based host responses 1(A): Age related factors within the host: To evaluate the effect of age on the onset and severity of reoviral arthritis in turkeys, the experimental turkey infection study was completed. Commercial turkey poults, derived from breeder hens vaccinated with autogenous vaccines and with known reovirus antibody endpoint titers, were orally inoculated with 4×106 TCID50 of TARV O'Neil at 1, 3, and 7 weeks of age (WOA) and euthanized at 3-, 7-, and 28-days post infection (dpi). Cloacal virus shedding, virus isolation from tendons, and virus migration to tendons was measured. The hock joints were processed to prepare slides and were kindly scored by Dr. Saad, Poultry Pathologist, University of Minnesota following the previously described scoring system (Sharafeldin et al., 2015). TARV induced weight gain suppression was pronounced when infected at 1 week of age compared with poults infected at 3 and 7 weeks of age. The average hock joint inflammation score was significantly higher compared to mock at 4 weeks post infection (wpi), when poults were infected at 1 and 3 weeks of age. The severity of inflammation in the hock joint is comparatively higher than age matched control birds when infected at either 1 week of age or 3 weeks of age. Also, cloacal viral shedding and virus isolation from tendons correlated with age dependent severity of inflammation of tendons associated with infection. Conclusively, maternally derived antibodies, derived from breeder hens vaccinated with autogenous vaccines, is likely insufficient to prevent same strain TARV infection and associated pathology at 4 weeks post infection. Additionally, the representative serum samples were also sent to Dr. Sunil Mor, South Dakota State University (SDSU) to run those samples on ELISA which demonstrated that the poults were seroconverted when poults are infected at 1, 3 or 7 weeks of age. Also, the samples are currently being processed for next generation sequencing for metataxonomic and transcriptomics. 1(B): Effect of naturally induced and passively acquired immunity: We will determine how pre-existing immunity or concurrent coinfection with the ubiquitous reovirus and TARV affects the onset and development of disease in SPF turkeys. For this experiment, hyper-immune reovirus anti-sera will be injected into the yolk sac of embryonating SPF turkey eggs. Therefore, hyperimmune serum (HIS) was produced in SPF turkeys by vaccination with TARV O'Neil and TERV MN1 inactivated whole virus vaccines adjuvanted with montanide. The poults were vaccinated at 5 weeks of age and booster doses were given at 8, 10 and 12 weeks of age. The poults for both groups, TARV O'Neil and TERV MN1 seroconverted at 5- and 6-weeks post vaccination and these sera will be used for passive immunization of eggs for the prospective antibody study.

Publications

• Type: Conference Papers and Presentations Status: Submitted Year Published: 2024 Citation: Saroj Khatiwada, John Ngunjiri, Patricia Boley, Kush Yadav1, Carolyn Lee1, Chang-Won Lee, Saad Gharaibeh, Gireesh Rajashekara, Scott P Kenney.Pathogenesis of reoviral arthritis in turkeys derived from vaccinated breeder hens. CRWAD, Jan 21-24, 2024

Progress 07/01/21 to 06/30/22

Outputs
Target Audience:Poultry industry including backyard poultry farmers; Goverment and state officials involved in control of animal diseases. Scientists in the field of infectious diseases, animal production, Ag-engineering, virology, bacteriology, immunology & vaccinology. Extension and outreach specialists. Changes/Problems:Drs. Chang-Won Lee and John Ngunjiri left Ohio State University, necessitating change in PI and co-PI to Gireesh Rajashekara and Scott Kenney. This has delayed the progress of the project. In addition, due to Covid related issues, hiring a graduate was delayed due to not getting visa on time. What opportunities for training and professional development has the project provided?One graduate student, two research assistantare currently working on this project and one Research Scientist worked on this project past year How have the results been disseminated to communities of interest?The results from this study are being disseminated through conferences, publications and meetings. What do you plan to do during the next reporting period to accomplish the goals?1) Viral, host, and intrinsic microbial factors contribute toward the onset, development, and severity of reovirus-associated arthritis in turkeys 2) Host/viral factors responsible for microbiota disruption 3) Impact of microbiota disruption on turkey performance 4) Role of microbiota in outbreak and severity of reoviral arthritis 5) Effect of microbiota disruption on TARV-induced disease 6) Develop microbiome-based interventions

Impacts
What was accomplished under these goals? An initial experimental turkey infection study as proposed in Table 1 was accomplished during this project year. Reoviral arthritis was reproducible in commercial and SPF turkeys. Disease was indicated by clinical manifestations and pathologic features such as staggered movement, dropped keel bone, swelling of hock joints, gastrocnemius, and digital flexor tendons, mild to severe valgus, distal tibiotarsal cartilage pitting and erosion, high gait scores, and weight gain depression. Turkey arthritis reovirus infection was associated with gut dysbiosis. Maternally-derived reovirus-specific antibodies in commercial turkeys were not protective against TARV-induced weight gain depression, cloacal virus shedding, or migration of TARV to hock joint tendons.Age-dependent resistance was observed regarding cloacal virus shedding and virus migration to the hock joint tendons at 4 weeks post-infection Samples are currently being processed for microscopic pathological evaluations, extraction of RNA for RNAseq transcriptional analysis and extraction of DNA for microbiome analysis.Data was presented with a poster at the Conference for Research Workers in Animal Diseases (CRWAD) meeting December 3-5th, 2021 Chicago Illinois.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Ngunjiri J., Lee, C. W. et al. Pathogenesis and control of emerging reoviral arthritis in turkeys. Conference of Research Workers in Animal Diseases, Chicago, IL, Dec 5, 2021 (poster)