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Author ORCID Identifier

https://orcid.org/0009-0000-2255-6366

Date Available

4-30-2027

Year of Publication

2026

Document Type

Doctoral Dissertation

Degree Name

Doctor of Philosophy (PhD)

College

Agriculture, Food and Environment

Department/School/Program

Veterinary Science

Faculty

Feng Li

Faculty

Emma Adam

Abstract

Despite a monovalent G3P[12] (‘G3’) vaccine being available for horses, equine rotavirus A (ERVA) is still the predominant infectious pathogen causing diarrhea in foals in the United States of America (U.S.). ERVA is classified into G genotypes based on the VP7 protein and P genotypes based on the VP4 protein. Two genotypes of ERVA, G3 and G14P[12] (‘G14’) are currently circulating in U.S. horses. Mares are immunized at 8, 9, and 10 months of gestation with the G3 vaccine and maternal antibodies are passively transferred to foals through colostrum. Outbreaks in foals from unvaccinated mares can be observed as early as 12 hours of age, while outbreaks in foals from vaccinated mares predominantly occur at later ages.

The first aim of our study was to determine how long virus neutralizing maternal antibodies from ERVA vaccinated dams persist in foals. For this aim, clinical samples including serum, milk, colostrum, and fecal swab samples were collected from 50 mare-foal pairs. Serum, colostrum, and milk samples were quantitatively tested for protective antibodies using a cell-based virus neutralization assay for both G3 and G14 viruses, while fecal swabs were analyzed by RT-qPCR for detection of ERVA genome. The absence of virus neutralizing antibodies (NAbs) in foal pre-nursing samples and the presence thereof after nursing confirmed that these NAbs are transferred through the colostrum. We also found that there is variation in the ratio of NAbs transferred between mares and their foals. Furthermore, results of our studies demonstrated a substantial cross-reactivity between G3 and G14 viruses in horses in that NAb titers against the G14 virus are approximately 2-4-fold lower than those against the homologous G3 virus. Both G3 and G14 NAb titers decreased steadily over time in foals with the lowest titers measured at approximately 4 months of age which is in line with when ERVA infections are observed in foals from vaccinated dams.

The second aim of our work was to determine if it is possible to increase NAb titers in foals through vaccination before they are vulnerable to ERVA infection. We immunized two foals with the commercially available vaccine (G3) at solely three months of age, seven foals at both two and three months of age, and two mock foals were vaccinated with saline buffer. The dams of these foals were not vaccinated during their gestation period. All pre-vaccination G3 and G14 NAb titers in this foal cohort were 256 or lower. Following vaccination, NAb titers in foals were increased up to 1024 against G3 and 512 against G14 viruses, respectively. Interestingly, NAb titers were stabilized in immunized foals, which contrasts with unvaccinated foals showing a rapid decline in NAb titers over time.

In the third aim of this project, we proposed to characterize the genetic diversity of current circulating genotypes in U.S. horses and determine whether the identified viruses behave different from each other in in vitro replication and antigenic property. A multidisciplinary approach including virus isolation, sequencing, genetic analysis, and antigenic characterization was utilized. Among 160 ERVA positive samples collected from 22 states, genotype-specific RT-PCR analysis showed 96 samples were positive for solely G3, 11 samples for G14, 52 samples for both G3 and G14, and 1 uncharacterized ERVA was identified. Thirty-three positive samples were sequenced using Illumina© MiSeq platform. Based on whole genome analysis, twenty unique viruses were identified. Based on amino acid sequences thirteen viruses were unique in genomic segment 4 (VP4) and 7 in segment 9 (VP7). Furthermore, extensive reassortment was detected among these viruses, indicating that co-infection of homologous and heterologous genotypes (G3 and G14) is common in horses, leading to the generation of new strains. Genetically different viruses were antigenically characterized using rabbit and equine sera. Despite no major antigenic differences observed between strains within the same genotype, rabbit serum, but not equine serum, distinguished the antigenic drift between G3 and G14 genotypes. Finally, experiments showed a genotype-dependent difference in replication kinetics with G14 viruses appearing to replicate slower than G3 viruses. This work revealed a novel host species-specific immunity against ERVA, which will be important for future rotavirus vaccine research.

Overall, these studies show that ERVA maternal antibodies decrease over time in foals which is in line with when most infections in foals from vaccinated dams are observed in the field study. NAbs generated in response to the G3 vaccine were cross-protective against G14, indicating that the current vaccine may sufficiently protect horses from both genotypes. Furthermore, it was shown that vaccination of foals is possible, however, the timepoint of vaccination and the pre-vaccination ERVA NAb titer is of influence on the immune response to vaccine antigens. Lastly, no isolates were identified that are antigenically different from each other within the G3 and within the G14 genotypes. However, viruses with different replication fitness were identified, indicating the potential emergence of evolutionary advanced rotaviruses with altered transmissibility in horses.

Despite the research presented in this dissertation that can offer novel insights to infection landscape of ERVA and vaccine-mediated protection in U.S. horses, challenge experiments involving vaccinated horses will shed light on the exact ERVA NAb titers needed to protect foals from infection and clinical disease. Furthermore, continued surveillance and isolation of future field strains from severely ill or diseased foals are important for the rapid identification of antigenically drifted strains so proper control and vaccination strategies can be implemented to protect foal health and mitigate economic losses to the equine industry. The development of a reverse genetic system would be useful for elucidating the impact of genetic mutations on viral antigenic property and determining the correlations of antibody-mediated protection.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2026.250b

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Archival

Funding Information

Maxwell and Muriel Gluck Fellowship in the Department of Veterinary Science (2021-2026)

Dr. Li’s William Robert Mills Endowed Chair Fund (2021-2026)

Zoetis

National Institute of Food and Agriculture project award no. 2025-67016-44998 from the United States Department of Agriculture

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Available for download on Friday, April 30, 2027

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