The study, published in Frontiers in Veterinary Science, was motivated by a specific clinical problem: myosin heavy chain myopathy, or MYHM, a recently described genetic disorder of Quarter Horses and related breeds such as Appaloosas and American Paint Horses. The condition stems from a single non-synonymous mutation, E321G, in the MYH1 gene, which encodes the myosin-type 2X heavy chain. Horses carrying the variant allele are susceptible to non-exertional rhabdomyolysis, immune-mediated myositis, and systemic calcinosis, syndromes that can produce muscle stiffness, weakness, myoglobinuria, and acute, severe muscle wasting. The variant allele is present in roughly 6.8 percent of American Quarter Horses, with the highest prevalence among reining, working cow, and halter performance lines.

Because the exact pathogenesis of immune-mediated myositis in these horses remains unclear, researchers have looked for environmental triggers. Early evidence suggested that clinical signs may be set off by exposure to an infectious agent or by vaccination itself. Some experts now recommend avoiding vaccination of horses carrying the mutation, or restricting them to only the most essential monovalent vaccines. What remains unknown is whether the muscle trauma caused by an intramuscular injection or the immune response provoked by the vaccine is the inciting event. If muscle damage from the needle is the culprit, delivering vaccines subcutaneously might reduce the risk of triggering disease in susceptible animals—a hypothesis the new study set out to test indirectly by comparing the two routes in healthy horses.

The research team, led by Zuzanna M. Pietras of Kansas State University with colleagues from Iowa State University, Washington State University, and the Virginia Maryland College of Veterinary Medicine, enrolled 17 systemically healthy adult mares from a university research and teaching herd. Sixteen were American Quarter Horses and one was a Thoroughbred. Their mean age was 14.1 years and their mean body weight 542 kilograms. Crucially, all horses tested negative for the MYH1 mutation, meaning the study evaluated the routes in animals without the genetic susceptibility. All of the mares had been vaccinated previously with a combination vaccine including West Nile virus and tetanus toxoid, with annual spring boosters for several years—a detail that would later complicate interpretation of the immune data.

The trial used a randomized, single-blind, two-arm, two-period design. On day 0, the mares received an inactivated West Nile virus vaccine; on day 28, they received a tetanus toxoid vaccine that is approved for subcutaneous use. Nine horses in group 1 were vaccinated subcutaneously in the neck with the West Nile vaccine and intramuscularly with tetanus toxoid, while eight horses in group 2 received the reverse assignment. Both injection routes targeted the same anatomical region: intramuscular injections went into the cervical serratus ventralis muscle, and subcutaneous injections into the tissue overlying that same muscle, avoiding the cervical vertebrae, nuchal ligament, and scapula. Injection sites were clipped, cleaned with isopropyl alcohol, and marked so that a blinded observer could monitor them consistently.

Antibody responses were tracked with a battery of assays performed at Cornell University’s Animal Health Diagnostic Center. West Nile virus IgM and IgG levels were measured with isotype-specific capture enzyme-linked immunosorbent assays, reported as a positive-to-negative ratio, and IgG was additionally assessed with a serum neutralization assay. Blood was collected on days 0, 28, and 56 for West Nile virus titers and on days 28 and 56 for tetanus titers. Local reactions were assessed daily for 72 hours after each injection, with swelling measured using calipers and pain scored on a four-point scale from no pain to moderate-to-severe pain in response to manual palpation. Serum amyloid A, a sensitive acute-phase protein that rises rapidly during systemic inflammation, was measured before and 24 hours after each vaccination.

The immunological results were equivocal in an instructive way. Serum neutralization titers against West Nile virus rose significantly by 28 days after vaccination regardless of route, with a p-value of 0.007, and no differences emerged between the subcutaneous and intramuscular groups. IgM levels were low at the start and did not change, indicating that no natural West Nile virus exposure had occurred before or during the study. However, nearly all of the horses began the trial with very high baseline anti-tetanus immunoglobulin titers and West Nile virus neutralization titers, many at or near the maximum values reportable by the diagnostic laboratory—almost certainly a legacy of their years of annual boosters. Tetanus titers did not increase after revaccination, and the authors caution that the statistically significant rise in West Nile virus titers may not be biologically meaningful given the high starting points. Definitive conclusions about whether subcutaneous vaccination produces immune responses equivalent to intramuscular vaccination therefore cannot be drawn from these data.

The adverse-effect findings, by contrast, were unambiguous. Injection site swelling was significantly more frequent and larger after subcutaneous administration of both vaccines. After the West Nile vaccine, eight of nine subcutaneously vaccinated horses developed visible swelling, with a median area of 8,467 square millimeters, compared with three of eight intramuscularly vaccinated horses, whose median swelling was essentially zero. After tetanus toxoid, every subcutaneously vaccinated horse developed swelling, versus four of nine after intramuscular injection. Pain on palpation followed the same pattern: very mild to moderate pain was detected in six of nine subcutaneously vaccinated horses after the West Nile vaccine and in five of eight after tetanus toxoid, while no pain was detected after intramuscular West Nile vaccination and only one horse reported pain after intramuscular tetanus vaccination. Seven of the nine horses that received the subcutaneous West Nile vaccine required systemic phenylbutazone and topical diclofenac cream because of significant swelling and pain; no horse required treatment after intramuscular administration of that vaccine, and none required treatment after tetanus toxoid by either route.

Systemic reactions were mild and largely route-independent. Two horses in each group developed a self-limiting fever of 38.7 to 38.8 degrees Celsius on the day after West Nile vaccination, resolving by day 2, and no horses developed fever after tetanus toxoid. Serum amyloid A concentrations rose after West Nile vaccination regardless of route, with no detectable difference between the groups, but remained below the detection threshold of 20 micrograms per milliliter after tetanus toxoid administration in all horses, suggesting that the tetanus vaccine does not provoke a significant systemic inflammatory response. No signs of abscess formation were observed at any injection site during the monitoring period.

The authors emphasize that the study’s implications for horses with the MYH1 mutation remain uncertain. The visible inflammation after subcutaneous injection was primarily confined to subcutaneous tissues, but inflammatory changes in the underlying muscle were not critically evaluated, and serum creatine kinase activities were not measured—so it is possible that the markedly greater local inflammatory response could actually be detrimental for genetically susceptible horses rather than protective. Other limitations include the small sample size, the restriction to mares, the use of previously vaccinated animals, and the inability to assess cellular immune responses. The researchers suggest that future work should use naïve, unvaccinated horses to compare humoral responses accurately, and should examine the influenza, equine herpesvirus 4, and strangles vaccines that have been more commonly implicated in triggering immune-mediated myositis. They also note that antibody measurements might eventually serve as an alternative to routine booster vaccination in horses prone to severe vaccine reactions, provided protective thresholds for West Nile virus and tetanus in horses can be definitively established. For now, the practical message is that subcutaneous vaccination in horses, at least with these two products, comes at the cost of substantial local reactions that many owners are likely to find unacceptable.

Subject of Research: Comparison of subcutaneous and intramuscular vaccination routes for antibody responses and adverse effects in horses, in the context of MYH1-related myopathy

Article Title: Humoral immune responses and adverse effects of inactivated West Nile virus and tetanus toxoid vaccines administered via the subcutaneous or intramuscular route in horses

Article References: Pietras, Z. M., Sanz, M. G., Pinnell, E., Rangel, A., & Sellon, D. C. (2026). Humoral immune responses and adverse effects of inactivated West Nile virus and tetanus toxoid vaccines administered via the subcutaneous or intramuscular route in horses. Frontiers in Veterinary Science, 13, Article 1931255. https://doi.org/10.3389/fvets.2026.1931255

Image Credits: AI Generated

DOI: 10.3389/fvets.2026.1931255

Keywords: horses, vaccination, subcutaneous injection, intramuscular injection, West Nile virus, tetanus toxoid, myosin heavy chain myopathy, immune-mediated myositis, MYH1 mutation, antibody titers, injection site reactions, serum amyloid A