Progress 07/01/20 to 06/30/24
Outputs
Target Audience:The target audiences reached during this project are equine veterinarians and veterinary scientists. Equine veterinarians were reached through personal communication via email and phone, and through formal presentations as indicated in the Publications section. Veterinarians have reached our team for case consultations through corresponding author details on publications, through referral by the New York State Animal Health Diagnostic Center Veterinary Support Services, through Dr. Tomlinson's work as an internist at Cornell Equine Animal Hospital and in her new position at the University of Pennsylvania New Bolton Center, and through Dr. Tomlinson's active presence on social media (Facebook Equine Vet-2-Vet page) answering queries related to this grant's subject material. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?* Research Associate/graduate student Dr. Joy Tomlinson: Dr. Tomlinson completed her graduate training under Dr. Van de Walle during the performance of this award. Largely based on her successful endeavors in this work, she recently obtained a faculty position as Associate Professor of Large Animal Medicine with a 45% research effort at University of Pennsylvania New Bolton Center and will continue in a track of research on equine viral hepatitis. * Graduate student Dr. Mason Jager: Dr. Jager completed his graduate training under Dr. Van de Walle during the course of this award. Based on his productivity in this area of research, he received an NIH Mentored Clinical Scientist Research Career Development Award (K08) award to further study the molecular aspects of EqPV-H replication. He is currently an Assistant Professor at Cornell University on a pre-tenure track that will become tenure track in the coming year. How have the results been disseminated to communities of interest?The results from our work have been presented at various virtual and in-person meetings and conferences with audiences from various scientific fields of virology research, immunology research, and veterinary research, as well as clinical veterinarians. In addition, the results have been informally discussed with other scientists and equine clinicians and have been formally published in open access journals. Based on the results of this study, Dr. Tomlinson is finalizing an updated version of the AAEP White Paper on equine viral hepatitis to inform veterinarians and horse owners on aspects of parvoviral disease, diagnosis, and biosecurity. What do you plan to do during the next reporting period to accomplish the goals?This is the final reporting period. No additional studies are planned. Data analysis and manuscript preparations are in their final stages. At least 3 additional publications are anticipated.
Impacts
What was accomplished under these goals?
Aim 1: Epidemiologic modeling of natural EqPV-H infection kinetics and transmission in horses. Investigation of the epidemiology of EqPV-H is still in its infancy. We are monitoring EqPV-H infection kinetics in various equine populations to better define incidence, and to use these data sets for epidemiologic modeling. Completion of aim 1 will provide us with a comprehensive picture of the epidemiology and routes of transmission of EqPV-H. In the final year, we have completed quarterly sample collection from multiple horse herds. A total of 144 horses had completed sampling of greater than one year duration and 350 horses were sampled at least once. We monitored serum viremia by qPCR and serology by LIPS. We found a 24% viremia prevalence in 2020, 29% in 2021, 53% in 2022, and 39% in 2023 so far, and remaining qPCR analysis is nearing completion. Seroprevalence has been slightly higher at 28%, 37%, 53%, and 52% respectively. We observed transmission and seroconversion in only 2 adult horses. Infection and seroconversion were observed in 9 foals that received seropositive anti-Rhodococcus equi plasma, despite those lots having tested PCR negative. An additional 2 foals that had received seronegative plasma became infected. These two foals were housed with or nearby the other 9 infected foals. We have observed that horses maintain low level viremia that fluctuates above and below the limit of detection of PCR. This is important information to guide the use of previously infected horses as blood product donors, since negative PCRs do not indicate complete clearance of the virus. The high rate of infection of foals treated with seropositive plasma suggests that these lots might contain virus below the limit of detection. Serology might be a better screening tool to improve safety of equine biologic products. Final analysis and publication will be completed in concert with the associated study regarding Equine hepacivirus (EqHV) epidemiology in the coming year. In addition, as part of the EqHV study, we have analyzed 50-200 sera from presumably health horses in New York, Florida, Virginia, and Colorado to better establish PCR and seroprevalence of both EqHV and EqPV-H in US horses, in addition to viral sequencing to evaluate viral diversity. This analysis is near completion and a publication is anticipated in the coming year. Aim 2: Characterizing the liver pathogenicity of EqPV-H to further establish its clinical significance and improve diagnostic measures. Although we have strong evidence that EqPV-H causes a form of acute hepatic necrosis in horses, the full spectrum of EqPV-H liver pathology has not been fully elucidated. This aim has been completed and resulted in two published manuscripts. One in the Veterinary Journal and one in Veterinary Pathology. Aim 3: Determine the immunopathogenesis of EqPV-H. We found that the onset of hepatitis coincides both with seroconversion and a decline in viremia, suggesting that immune control of the infection might be associated with hepatic injury. We will assess the immune responses during experimental EqPV-H infections by performing (i) transcriptomic immune profiling of PBMC and liver biopsies using RNA deep sequencing, (ii) serology to determine kinetics in antibody levels in serum samples, and (iii) immunohistochemical analysis of immune cells in the liver. Completion of aim 3 will provide us with information regarding the involvement of the immune system in liver damage, and importantly, will be the necessary first step towards rationally-based vaccine design. We have completed the in vivo experimental infections. We observed subclinical disease in most, however 3 horses showed clinical signs associated with their hepatitis, with two horses moderately-severely affected and requiring hospitalization. Interestingly, colic was observed in 3 horses associated with the peak hepatitis and without any GI condition identified. All horses recovered. We assessed the immune responses during these experimental EqPV-H infections by performing (i) transcriptomic immune profiling of PBMC and liver biopsies using RNA deep sequencing, (ii) serology to determine kinetics in antibody levels in serum samples, (iii) immunohistochemical analysis of immune cells in the liver, and (iv) spatial transcriptomic analysis of the liver over the course of infection for 2 horses. All data have been collected and final analysis is ongoing. Publication is anticipated in the coming year.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Joy Tomlinson. Naturally acquired equine parvovirus-hepatitis is associated with a wide range of hepatic lesions in horses. CRWAD, Jan 2024, Chicago, IL.
|
Progress 07/01/22 to 06/30/23
Outputs
Target Audience:The target audiences reached during this reporting period are equine veterinarians and veterinary scientists. Equine veterinarians were reached through personal communication via email and phone, and through formal presentations as indicated in the Publications section. Veterinarians have reached our team for case consultations through corresponding author details on publications, through referral by the New York State Animal Health Diagnostic Center Veterinary Support Services, through Dr. Tomlinson's work as an internist at Cornell Equine Animal Hospital and her active presence on social media (Facebook Equine Vet-2-Vet page) answering queries related to this grant's subject material., as well through Dr. Mason Jager as an anatomic pathologist receiving liver tissue samples for analysis. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Our Research Associate/graduate student, Dr. Joy Tomlinson, and our graduate student, Dr. Mason Jager, both successfully graduated and obtained their PhD in 2021 and 2022, respectively. Dr. Mason moved up in a position as Assistant Research Professor at Cornell, building out his own independent lab and research activities and Dr. Joy Tomlinson recently received an offer for a faculty position at University of Pennsylvania, which will allow her to start her independent lab and research as well. How have the results been disseminated to communities of interest?The results from our work have been presented at various virtual and in-person meetings and conferences with audiences from various scientific fields of virology research, immunology research, and veterinary research, as well as clinical veterinarians. In addition, the results have been informally discussed with other scientists and equine clinicians and have been received with great enthusiasm by scientists in the field. The results have been formally published in open access journals. What do you plan to do during the next reporting period to accomplish the goals?During the coming year, which is the final year of the project, we will continue to collect and process quarterly serum samples from horses for Aim 1. Aim 2, is complete. For Aim 3, the in vivo aspect is complete and we will finalize sample analysis and prepare a manuscript for publication.
Impacts
What was accomplished under these goals?
Our long-term goal is to reduce and/or prevent transmission and disease caused by a novel equine parvovirus, named equine parvovirus hepatitis (EqPV-H). EqPV-H is strongly associated with Theiler's disease, a highly fatal syndrome of fulminant hepatitis in horses. Improving our understanding of the epidemiology and pathogenic mechanisms of this novel equine parvovirus will provide a strong basis for improved management strategies as well as future vaccine development and/or therapeutic interventions. Aim 1: Epidemiologic modeling of natural EqPV-H infection kinetics and transmission in horses. In the third year, we have continued quarterly sample collection from 120 horses. We monitored serum viremia by qPCR and serology. The total population was 24% viremia prevalence in 2020, 29% in 2021, 53% in 2022, and 39% in 2023 so far. Seroprevalence has been slightly higher at 28%, 37%, 53%, and 47% respectively. No transmission has been observed except within a herd of 11 horses that were experimentally inoculated with EqPV-H intranasally. It appears that 5 of those horses were infected by the experimental inoculation and 5 by transmission from the others in the herd. That transmission occurred in early spring before the activity of insect vectors. In the third year we added a couple additional herds for quarterly monitoring in the aim of capturing transmission events. Although not all the samples are analyzed, we have not yet observed natural transmission. We also have observed that horses maintain low level viremia that fluctuates above and below the limit of detection of PCR. This is important information to guide the use of previously infected horses as blood product donors, since negative PCRs do not indicate complete clearance of the virus. Serology could be a better screening tool. An article describing the experimental transmission study was published in 2022. Eleven horses were inoculated orally, after which no transmission occurred. Those horses were then inoculated nasally, after which 10 of 11 horses became infected at a very delayed incubation period of 6 to > 12 weeks. This demonstrates that biosecurity including physical separation of infected and naïve horses, and cleaning of the environment, could be important in reducing viral transmission in herds, although the long incubation period suggests such efforts might be impractical. Aim 2: Characterizing the liver pathogenicity of EqPV-H to further establish its clinical significance and improve diagnostic measures. Although we have strong evidence that EqPV-H causes a form of acute hepatic necrosis in horses, the full spectrum of EqPV-H liver pathology has not been fully elucidated. To address this, we analyzed EqPV-H infection and pathology in New York State racehorses and performed a retrospective analysis of banked clinical liver samples. For the racehorse study, DNA was extracted from archived formalin-fixed, paraffin-embedded liver tissues. A total of 191 liver samples from horses between 2 to 13 years old were evaluated. Forty-two samples were PCR positive (22%). Of those, 31 samples had positive viral nucleic acid hybridization in hepatocytes with 11 samples showing positive hybridization in necrotic hepatocytes associated with inflammatory cells, indicating active hepatitis. Both individual hepatocyte necrosis and hepatitis were positively associated with EqPV-H detection (p < .0001 and p = .0005, respectively). These findings indicate that presence of EqPV-H in the liver and parvoviral-associated hepatitis are prevalent in racehorses from New York racetracks, thus warranting additional studies examining potential associations between EqPV-H infection and racehorse performance. This manuscript is now published in the Virology Journal. To determine whether EqPV-H is associated with other clinical cases of liver disease in horses beyond the classic Theiler's disease, we performed a retrospective study of 98 banked liver biopsies from clinical cases of hepatitis. EqPV-H nucleic acid was detected in 48% (n=47) of samples. The most common histologic features of EqPV-H-positive samples included individual hepatocyte necrosis (n=40, 85%), lobular infiltrates (n=38, 80%), portal infiltrates (n=35, 74.4%), and ductular reaction (n=33, 70%). Fibrosis, portal edema, and neutrophilic infiltrates were negatively associated with the likelihood of EqPV-H-induced pathology. Portal infiltrate, and centrilobular necrosis were positively associated with the likelihood of EqPV-H-induced pathology. We also looked at the diagnostic patterns that were identified in the samples (bridging fibrosis, cholangiohepatitis, cholangitis, hepatic necrosis, lobular hepatitis, megalocytosis, portal hepatitis, toxic hepatopathy). Histologic diagnoses of hepatic necrosis and toxic hepatopathy were positively associated with EqPV-H infection, while cholangiohepatiits was negatively associated with parvoviral infection. Interestingly, EqPV-H was observed in two cases of hepatitis in foals and one case of lymphoma in the liver. To the best of our knowledge, this is the first report of EqPV-H-associated hepatitis in foals. In summary, this study demonstrates that EqPV-H is common in a variety of liver pathologies and should be considered as a differential diagnosis in cases of hepatitis other than Theiler's disease. This manuscript has been accepted in Veterinary Pathology and is currently under editorial revisions. Additionally, we have found that ISH hybridization signal increased proportionally to the severity of disease and could be used to distinguish between pathologic/active parvoviral infection vs. quiescent chronic infection that is an incidental finding. Based on our work, the New York State Animal Health Diagnostic Center has added a new diagnostic test of ISH for EqPV-H to their list of services. These slides are reviewed by our team member, Dr. Mason Jager, both improving diagnostic capabilities, and serving as a ready source of samples to improve our research and understanding of the disease. We have seen a significant rise in clinical submissions for ISH testing in the last year, which has greatly improved our diagnostic capabilities. The results of our retrospective testing have informed our clinical recommendations. For example, a horse died of acute hepatitis recently. The histopathology was not clearly an overt Theiler's disease case and was interpreted as suspected toxic hepatopathy. Based on our previous findings, we recommended ISH testing. ISH was performed and identified EqPV-H as a major cause of the horse's hepatitis. Aim 3: Determine the immunopathogenesis of EqPV-H. We found that the onset of hepatitis coincides both with seroconversion and a decline in viremia, suggesting that immune control of the infection might be associated with hepatic injury. We will assess the immune responses during experimental EqPV-H infections by performing (i) transcriptomic immune profiling of PBMC and liver biopsies using RNA deep sequencing, (ii) serology to determine kinetics in antibody levels in serum samples, and (iii) immunohistochemical analysis of immune cells in the liver. In the third year, we have completed the in vivo experimental infections. We have observed subclinical disease in most, however 3 horses showed clinical signs associated with their hepatitis, with two horses moderately-severely affected and requiring hospitalization. Interestingly, colic was observed in 3 horses associated with the peak hepatitis and without any gastro-intestinal condition identified. All horses recovered. Sample analysis will be finalized in the coming year.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Department of Biomedical Sciences faculty candidate research seminar, July 2021, Ithaca, NY. Mason Jager. Unraveling the pathology and replication dynamics of equine parvovirus-hepatitis.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
American College of Veterinary Pathology, Boston, MA. Mason Jager. Naturally acquired equine parvovirus-hepatitis is associated with a wide range of hepatic lesions in horses.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Jager MC, Tomlinson JE, Henry CE, Fahey MJ, Van de Walle GR. Prevalence and pathology of equine parvovirus-hepatitis in racehorses from New York racetracks. Virology Journal (2022) 19:175-184.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Tomlinson JE, Van de Walle GR. Nasal transmission of equine parvovirus-hepatitis. (2022) 36:2238-2244.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
CRWAD, Jan 2023, Chicago, IL. Joy Tomlinson. Prevalence and pathology of Equine parvovirus-hepatitis in racehorses from New York racetracks.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Department of Clinical Studies research seminar, Feb 2023, New Bolton Center, Kennett Square, PA. Joy Tomlinson. Immunopathology of equine viral hepatitis.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2023
Citation:
Jager MC, Choi E, Tomlinson JE, Van de Walle GR. Naturally acquired equine parvovirus-hepatitis is associated with a wide range of hepatic lesions in horses. Veterinary pathology.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Veterinary Science Seminar, Aug 2023, University of Kentucky Veterinary Diagnostic Laboratory, Lexington, KY. Mason Jager. Equine Viral Hepatitis: More Common that we Thought.
|
Progress 07/01/21 to 06/30/22
Outputs
Target Audience:The target audiences reached during this reporting period are equine veterinarians and veterinary scientists. Equine veterinarians were reached through personal communication via email and phone, and through formal presentations as indicated in the Publications section. Veterinarians have reached our team for case consultations through corresponding author details on publications, through referral by the New York State Animal Health Diagnostic Center Veterinary Support Services, through Dr. Tomlinson's work as an internist at Cornell Equine Animal Hospital, and active presence on social media (Facebook Equine Vet-2-Vet page) answering queries related to this grant's subject material. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?* Research Associate/graduate student Dr. Joy Tomlinson: graduate training under Dr. Van de Walle. Dr. Tomlinson has been doing the in vivo transmission studies, as well as collecting regular blood samples for the proposed epidemiology modeling. Dr Tomlinson is well trained in various lab techniques, including flow cytometry. Working on the project greatly strengthens her competences in optimizing protocols as well as her independence to establishing new protocols, realizing innovative ideas and presenting data to the scientific community. Further, the project allowed to develop new analytical skills to interpret data. The work broadens her skills related to epidemiology, immunology, and virology. Based on her productivity in these studies, Dr. Tomlinson completed her PhD in December 2021 and has submitted her portfolio for promotion to Senior Research Associate in August 2022. * Graduate student Dr. Mason Jager: graduate training under Dr. Van de Walle. Dr. Jager has been involved in collecting and analyzing cases for the proposed retrospective studies. Dr. Jager is well trained in anatomic pathology (board-certified). Working on the project greatly strengthens his competences in optimizing protocols as well as his independence to establishing new protocols, realizing innovative ideas and presenting data to the scientific community. Further, the project allowed him to develop new analytical skills to interpret data. The work broadens his skills related to molecular virology and in situ hybridization (ISH), and has prompted him to submit an NIH Mentored Clinical Scientist Research Career Development Award (K08) award based on the molecular aspects of EqPV-H replication. His resubmission application, submitted in November 2021 to NIAID received a top score and he recently received his notice of award. His accomplishments on this project have led to completion of his PhD in May 2022 and promotion to tenure track faculty. How have the results been disseminated to communities of interest?The results from our work have been presented at various virtual and in-person meetings and conferences with audiences from various scientific fields of virology research, immunology research, and veterinary research, as well as clinical veterinarians. In addition, the results have been informally discussed with other scientists and equine clinicians and have been received with great enthusiasm by scientists in the field. What do you plan to do during the next reporting period to accomplish the goals?During the coming year, we will continue to collect and process quarterly serum samples from horses for Aim 1. For Aim 2, we will finalize revisions on the manuscript describing the findings in racehorses, and will submit the manuscript of the case-control study of clinical cases. We will also enroll the remaining horses by donation or purchase for the Aim 3 experimental inoculations and immunopathogenesis studies. We will use biopsies from the horses in the transmission study completed last year for spatial transcriptomic analysis. The spatial transcriptomic analysis will be used to assess pathogenic mechanisms in parvoviral hepatitis and to guide additional targeted analysis in the Aim 3 investigation.
Impacts
What was accomplished under these goals?
Our long-term goal is to reduce and/or prevent transmission and disease caused by a novel equine parvovirus, named equine parvovirus hepatitis (EqPV-H). EqPV-H is strongly associated with Theiler's disease, a highly fatal syndrome of fulminant hepatitis in horses. Improving our understanding of the epidemiology and pathogenic mechanisms of this novel equine parvovirus will provide a strong basis for improved management strategies as well as future vaccine development and/or therapeutic interventions. Aim 1: Epidemiologic modeling of natural EqPV-H infection kinetics and transmission in horses. Investigation of the epidemiology of EqPV-H is ongoing. We are monitoring EqPV-H infection kinetics in various equine populations to better define incidence, and to use these data sets for epidemiologic modeling. Completion of aim 1 will provide us with a comprehensive picture of the epidemiology and routes of transmission of EqPV-H. In the second year, we have continued quarterly sample collection from 120 horses, and analysis of 44 paired mare-foal samples. So far, viremia prevalence was 24%, 29%, and 16%, in year 2020, 2021, and so far, 2022. No transmission has been observed except within a herd of 11 horses that were experimentally inoculated with EqPV-H intranasally. It appears that 5 of them were infected by the experimental inoculation and 5 by transmission from the others in the herd. The latter occurred in early spring before the activity of insect vectors. We also have observed that horses maintain low level viremia that fluctuates above and below the limit of detection of PCR. This is important information to guide the use of previously infected horses as blood product donors, since negative PCRs do not indicate complete clearance of the virus. In the second year, we also completed the analysis and manuscript preparation describing the experimental transmission study. Eleven horses were inoculated orally, after which no transmission occurred. Those horses were then inoculated nasally, after which 10 of 11 horses became infected at a very delayed incubation period of 6 to > 12 weeks. This demonstrates that biosecurity including physical separation of infected and naïve horses, and cleaning of the environment, could be important in reducing viral transmission in herds, although the long incubation period suggests such efforts might be impractical. Aim 2: Characterizing the liver pathogenicity of EqPV-H to further establish its clinical significance and improve diagnostic measures. Although we have strong evidence that EqPV-H causes a form of acute hepatic necrosis in horses, the full spectrum of EqPV-H liver pathology has not been fully elucidated. To address this, we analyzed EqPV-H infection and pathology in New York State racehorses and performed a case-controlled retrospective analysis of banked clinical liver samples. In parallel, we will analyze the liver biopsy samples from the experimentally infected horses to better define the histopathologic characteristics of EqPV-H infections. Completion of aim 2 will provide us with information regarding the histopathological liver characteristics of EqPV-H, to clearly define the clinical significance of this virus, and to improve diagnostic evaluation of liver biopsies. To determine the prevalence and pathogenicity of EqPV-H infection at New York racetracks, DNA was extracted from archived formalin-fixed, paraffin-embedded liver tissues from racehorses. A total of 191 liver samples from horses between 2 to 13 years old were evaluated. Forty-two samples were PCR positive (22%). Of those, 31 samples had positive viral nucleic acid hybridization in hepatocytes with 11 samples showing positive hybridization in necrotic hepatocytes associated with inflammatory cells, indicating active hepatitis. Both individual hepatocyte necrosis and hepatitis were positively associated with EqPV-H detection (p < .0001 and p = .0005, respectively). These findings indicate that presence of EqPV-H in the liver and parvoviral-associated hepatitis are prevalent in racehorses from New York racetracks, thus warranting additional studies examining potential associations between EqPV-H infection and racehorse performance. To determine whether EqPV-H is associated with other clinical cases of liver disease in horses beyond the classic Theiler's disease, we performed a retrospective study of 98 banked liver biopsies from clinical cases of hepatitis. EqPV-H nucleic acid was detected in 48% (n=47) of samples. The most common histologic features of EqPV-H-positive samples included individual hepatocyte necrosis (n=40, 85%), lobular infiltrates (n=38, 80%), portal infiltrates (n=35, 74.4%), and ductular reaction (n=33, 70%). Fibrosis, portal edema, and neutrophilic infiltrates were negatively associated with the likelihood of EqPV-H-induced pathology. Portal infiltrate, and centrilobular necrosis were positively associated with the likelihood of EqPV-H-induced pathology. We also evaluated diagnostic patterns that were identified in the samples (bridging fibrosis, cholangiohepatitis, cholangitis, hepatic necrosis, lobular hepatitis, megalocytosis, portal hepatitis, toxic hepatopathy). Histologic diagnoses of hepatic necrosis and toxic hepatopathy were positively associated with EqPV-H infection, while cholangiohepatitis was negatively associated with parvoviral infection. Interestingly, EqPV-H was observed in two cases of hepatitis in foals and one case of lymphoma in the liver. To the best of our knowledge, this is the first report of EqPV-H-associated hepatitis in foals. Our study demonstrates that EqPV-H is common in a variety of liver pathologies and should be considered as a differential diagnosis in cases of hepatitis other than Theiler's disease. Additionally, we found that ISH hybridization signal increased proportionally to the severity of disease and could be used to distinguish between pathologic/active parvoviral infection versus quiescent chronic infection that is an incidental finding. Based on our work, the New York State Animal Health Diagnostic Center has added a new diagnostic test of EqPV-H ISH to their list of services. These slides are reviewed by our team member, Dr. Mason Jager, both improving diagnostic capabilities, and serving as a ready source of samples to improve our research and understanding of the disease. We have seen a significant rise in clinical submissions for ISH testing in the last year, which has greatly improved our diagnostic capabilities. The results of our retrospective testing have informed our clinical recommendations. Aim 3: Determine the immunopathogenesis of EqPV-H. We found that the onset of hepatitis coincides both with seroconversion and a decline in viremia, suggesting that immune control of the infection might be associated with hepatic injury. We will assess the immune responses during experimental EqPV-H infections by performing (i) transcriptomic immune profiling of PBMC and liver biopsies using RNA deep sequencing, (ii) serology to determine kinetics in antibody levels in serum samples, and (iii) immunohistochemical analysis of immune cells in the liver. Completion of aim 3 will provide us with information regarding the involvement of the immune system in liver damage, and importantly, will be the necessary first step towards rationally-based vaccine design. In the first year of the project, we developed an experimental design plan with NYU to perform spatial transcriptomics on liver biopsies from experimentally infected horses. The remainder of this aim will be performed on experimentally infected horses using intravenous inoculation. By now, we have enrolled 5 of the target 10 horses and have completed sample collections from 3 of those. We have identified biopsies from two of those horses that will be appropriate for spatial transcriptomics and are arranging for the transcriptomic testing.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Jager MC, Tomlinson JE, Lopez-Astacio RA, Parrish CR, Van de Walle GR. (2021). Small but mighty: old and new parvoviruses of veterinary significance. Virol J. 18: 210.
- Type:
Other
Status:
Published
Year Published:
2021
Citation:
Joy Tomlinson. The Equine Hepatitis Viruses disease and diagnosis. Ohio State University College of Veterinary Medicine Equine Research Group Meeting and Seminar Series, June 2021, via zoom.
- Type:
Other
Status:
Published
Year Published:
2021
Citation:
Joy Tomlinson. Equine viral hepatitis: clinical conditions and epidemiology. American College of Veterinary Internal Medicine, Austin, TX.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Joy Tomlinson. The equine hepatitis viruses. American Association of Equine Practitioners, December 2021, Nashville, TN.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Joy Tomlinson, Thomas Divers, Gerlinde Van de Walle. Equine parvovirus-hepatitis transmission. 102th Annual Meeting of CRWAD.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2021
Citation:
Joy Tomlinson. Tropism, transmission, and immunopathology of equine hepatitis viruses.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2022
Citation:
Mason Jager. Pathology and Pathogenesis of equine parvovirus-hepatitis.
|
Progress 07/01/20 to 06/30/21
Outputs
Target Audience:The target audiences reached during this reporting period are equine veterinarians, veterinary scientists, and regulatory agencies (USDA). Equine veterinarians were reached through personal communication via email and phone, and through formal presentations as indicated in the Publications section. Veterinarians have reached out to our team for case consultations through corresponding author details on publications, through referral by the New York State Animal Health Diagnostic Center Veterinary Support Services, through Dr. Tomlinson's work as an internist at Cornell Equine Animal Hospital, and through Dr. Tomlinson's active presence on social media (Facebook Equine Vet-2-Vet page) answering queries related to this grant's subject material. Finally, Drs. Tomlinson and Van de Walle have also consulted in virtual meetings with agents at the USDA regarding equine parvovirus testing methods and requirements. Changes/Problems:Despite COVID disruptions, we were able to progress with the project on schedule. We were surprised to find that all 11 horses in the transmission study were successfully inoculated intranasally. This means there will be a delay in Aim 3 while we acquire new horses for that aim. We expect that this remains feasible. What opportunities for training and professional development has the project provided?* Research Associate/graduate student Dr. Joy Tomlinson: graduate training under Dr. Van de Walle. Dr. Tomlinson has been doing the in vivo transmission studies, as well as collecting regular blood samples for the proposed epidemiology modeling. Dr Tomlinson is well trained in various lab techniques, including flow cytometry. Working on the project greatly strengthens her competences in optimizing protocols as well as her independence to establishing new protocols, realizing innovative ideas and presenting data to the scientific community. Further, the project allowed to develop new analytical skills to interpret data. The work broadens her skills related to epidemiology, immunology, and virology. Dr. Tomlinson has also been awarded for her work as follows: American Association of Equine Practitioners 2021 Research Award - established to highlight the value of equine research and give recognition to an individual who has recently completed research that has or will make a significant impact on the diagnosis, treatment or prevention of equine disease. Phi Zeta Alpha Chapter manuscript competition 2021, winner of the basic science category for "Tropism, pathology, and transmission of equine parvovirus-hepatitis." Bicknese Family Prize 2020 - to support an outstanding female scientist-in-training at the Baker Institute for Animal Health for their research activities. * Graduate student Dr. Mason Jager: graduate training under Dr. Van de Walle. Dr. Jager has been involved in collecting and analyzing cases for the proposed retrospective studies. Dr. Jager is well trained in anatomic pathology (board-certified). Working on the project greatly strengthens his competences in optimizing protocols as well as his independence to establishing new protocols, realizing innovative ideas and presenting data to the scientific community. Further, the project allowed him to develop new analytical skills to interpret data. The work broadens his skills related to molecular virology and in situ hybridization (ISH), and has prompted him to submit an NIH Mentored Clinical Scientist Research Career Development Award (K08) award based on the molecular aspects of EqPV-H replication. His application, submitted in November 2020 to NIAID was not funded upon first submission. How have the results been disseminated to communities of interest?The results from our work have been presented at various virtual meetings and conferences with audiences from various scientific fields of virology research, immunology research, and veterinary research, as well as clinical veterinarians. In addition, the results have been informally discussed with other scientists and equine clinicians and have been received with great enthusiasm by scientists in the field. What do you plan to do during the next reporting period to accomplish the goals?During the coming year, we will continue to collect and process quarterly serum samples from horses for Aim 1. For Aim 2, we expect to submit a manuscript describing the findings in racehorses, as we continue the analysis of the case-control study of clinical cases. We have established a collaboration with UC Davis who will be sending us 50 additional samples. We will also enroll horses by donation or purchase for the Aim 3 experimental inoculations and immunopathogenesis studies. We expect to enroll at least 5 horses in the coming year. We will use biopsies from the horses in the transmission study completed this year for spatial transcriptomic analysis. This will be used to assess pathogenic mechanisms in parvoviral hepatitis and to guide additional targeted analysis in the Aim 3 investigation.
Impacts
What was accomplished under these goals?
Our long-term goal is to reduce and/or prevent transmission and disease caused by a novel equine parvovirus, named equine parvovirus hepatitis (EqPV-H). EqPV-H is strongly associated with Theiler's disease, a highly fatal syndrome of fulminant hepatitis in horses. Improving our understanding of the epidemiology and pathogenic mechanisms of this novel equine parvovirus will provide a strong basis for improved management strategies as well as future vaccine development and/or therapeutic interventions. EqPV-H was known to be transmitted by veterinary administration of equine origin blood products, but it was unknown how it is transmitted between horses in a natural setting. In the past year we have determined that EqPV-H can be transmitted nasally. This is essential information indicating that isolation of acutely infected individuals could be used to reduce viral transmission and thus, reduce the risk of Theiler's disease. Aim 1: Epidemiologic modeling of natural EqPV-H infection kinetics and transmission in horses. Investigation of the epidemiology of EqPV-H is still in its infancy. We propose to monitor EqPV-H infection kinetics in various equine populations to better define incidence and to use these data sets for epidemiologic modeling. Completion of aim 1 will provide us with a comprehensive picture of the epidemiology and routes of transmission of EqPV-H. In the first year, we have completed quarterly sample collection from 100 horses, and 41 paired mare-foal samples. We monitored serum viremia by qPCR and have serology in process. We have also identified two herds of ~15-30 horses each that have 20 years of banked serum that we have access to for PCR and serology testing. So far, we have PCR-processed the quarterly samples from the ~100 horses collected this year. The total population started with 31% viremia prevalence in the second quarter of 2020, which has fallen to 15% a year later. We have observed that horses maintain low level viremia that fluctuates above and below the limit of detection of PCR. This is important information to guide the use of previously infected horses as blood product donors, since negative PCRs do not indicate complete clearance of the virus. Moreover, we have completed the experimental transmission study. Eleven horses were inoculated orally, after which no transmission occurred. Those horses were then inoculated nasally, after which all horses became infected at a very delayed incubation period of 6 to > 11 weeks. This demonstrates that biosecurity including physical separation of infected and naïve horses, and cleaning of the environment, will likely be important in reducing viral transmission in herds. Aim 2: Characterizing the liver pathogenicity of EqPV-H to further establish its clinical significance and improve diagnostic measures. Although we have strong evidence that EqPV-H causes a form of acute hepatic necrosis in horses, the full spectrum of EqPV-H liver pathology has not been fully elucidated. To address this, we will perform a case-controlled retrospective analysis of banked clinical liver samples. In parallel, we will analyze the liver biopsy samples from the experimentally infected horses to better define the histopathologic characteristics of EqPV-H infections. Completion of aim 2 will provide us with information regarding the histopathological liver characteristics of EqPV-H, to clearly define the clinical significance of this virus, and to improve diagnostic evaluation of liver biopsies. In this year, we have completed PCR and in situ hybridization (ISH) of 197 liver biopsies from New York State Thoroughbred racehorses. We have found a similar prevalence as the general population at 23%, with evidence of active pathology in 20% of ISH-positive cases (4% total). We have also identified and reviewed 55 clinical biopsies from 5 categories of liver disease. We found that ISH hybridization signal increased proportionally to the severity of disease and could be used to distinguish between pathologic/active parvoviral infection versus quiescent chronic infection that is an incidental finding. Additionally, 6/20 (30%) of lobular hepatitis cases were positive for EqPV-H, while all cases of cholangiohepatitis are EqPV-H negative. Interestingly, 5/10 (50%) of cases with megalocytosis, a feature often associated with pyrrolizidine alkaloid toxicity, were positive for EqPV-H. Based on our work, the New York State Animal Health Diagnostic Center has added a new diagnostic test for EqPV-H, based on ISH, to their list of services, which represents a change of action. These slides are reviewed by our team member and anatomic pathologist, Dr. Mason Jager, both improving diagnostic capabilities, and serving as a ready source of samples to improve our research and understanding of the disease. Aim 3: Determine the immunopathogenesis of EqPV-H. We found that the onset of hepatitis coincides both with seroconversion and a decline in viremia, suggesting that immune control of the infection might be associated with hepatic injury. We will assess the immune responses during experimental EqPV-H infections by performing (i) transcriptomic immune profiling of PBMC and liver biopsies using RNA deep sequencing, (ii) serology to determine kinetics in antibody levels in serum samples, and (iii) immunohistochemical analysis of immune cells in the liver. Completion of aim 3 will provide us with information regarding the involvement of the immune system in liver damage, and importantly, will be the necessary first step towards rationally-based vaccine design. In the first year of the project, we have developed an experimental design plan with NYU to perform spatial transcriptomics on liver biopsies from experimentally infected horses. The remainder of this aim will be performed on experimentally infected horses using intravenous inoculation. Because the transmission aim showed 100% transmission rate, we need to source an additional 10 horses for this experiment. We have 1 horse recently donated for this purpose. These horses will be enrolled and this aim completed over the next 2 years.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Harry M. Zweig Memorial Fund for Equine Research 2020 Virtual Faculty and Trainee Presentations, November 2020, Ithaca, NY. Joy Tomlinson. Equine hepatitis viruses: where are we now?
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Cornell Biomedical and Biological Sciences (BBS) Work in Progress, April 2021 Ithaca, NY. Mason Jager. The Replication Dynamics of Equine Parvovirus-H and its Role in Disease Pathogenesis.
- Type:
Other
Status:
Published
Year Published:
2021
Citation:
The Bicknese Family Prize Awardee presentation, May 2021, Ithaca, NY. Joy Tomlinson. Equine hepatitis viruses.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
American Society of Virology Meeting, Satellite Symposium on Viral Pathogenesis & Prevention in Animals, July 2021, virtual. Gerlinde Van de Walle. Pathogenesis of liver-tropic viruses in horses: a tale of trial and error.
- Type:
Other
Status:
Published
Year Published:
2021
Citation:
Ohio State University College of Veterinary Medicine Equine Research Group Meeting and Seminar Series, June 2021, virtual. Joy Tomlinson. The Equine Hepatitis Viruses disease and diagnosis.
- Type:
Other
Status:
Published
Year Published:
2021
Citation:
Department of Microbiology and Immunology Post-doctoral research seminar, May 2021, Ithaca, NY. Joy Tomlinson. Immune responses after acute equine hepacivirus infection.
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