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Effect of Long-Term Freezing on Indirect Fluorescent Antibody Titers for the Diagnosis of Equine Protozoal Myeloencephalitis.
Abstract: Long-term freezing storage can alter the stability of proteins, thereby compromising accurate determination of indirect fluorescent antibody test (IFAT) titers that support the diagnosis of equine protozoal myeloencephalitis. Objective: Assess the effect of long-term storage at -80°C on IFAT against S. neurona and N. hughesi in equine serum and cerebrospinal fluid (CSF). Methods: Paired serum and CSF (n = 46), and serum only (n = 25) samples. Methods: Prospective study of samples stored 6-12, 13-18, and 19-24 months. Comparing antibody titers across time points, McNemar and Wilcoxon rank tests were used for statistical analysis. Results: After long-term freezing storage for 19-24 months, N. hughesi IFAT results for CSF were significantly different compared with their fresh-testing time point (p = 0.04), but a similar difference was not observed for serum samples. No statistical differences were observed for serum or CSF samples tested for S. neurona after 6-12, 13-18, or 19-24 months compared with their original fresh testing results. However, the serum to CSF ratio cutoff of ≤ 64 in support of disease changed in 41% (19 of 46) of paired samples. Conclusions: Freezing samples at -80°C for more than 18 months might alter test results, leading to possible changes in serum to CSF ratio calculations and affecting the interpretation of results for clinical and research purposes.
© 2025 The Author(s). Journal of Veterinary Internal Medicine published by Wiley Periodicals LLC on behalf of American College of Veterinary Internal Medicine.
Publication Date: 2025-08-30 PubMed ID: 40873183PubMed Central: PMC12391721DOI: 10.1111/jvim.70225Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
- Antibodies
- Cerebrospinal Fluid
- Clinical Study
- Diagnosis
- Disease Diagnosis
- Disease Management
- Equine Diseases
- Equine Health
- Equine Protozoal Myeloencephalitis
- Freezing Technique
- Immunofluorescence Assay
- Immunology
- Laboratory Methods
- Protozoa
- Serology
- Serum
- Statistical Analysis
- Veterinary Medicine
- Veterinary Research
- Veterinary Science
Summary
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Overview
- This study investigates how long-term freezing at -80°C affects the accuracy of indirect fluorescent antibody test (IFAT) titers used to diagnose equine protozoal myeloencephalitis (EPM) by examining serum and cerebrospinal fluid (CSF) samples over periods up to 24 months.
Background
- Equine protozoal myeloencephalitis (EPM) is a neurological disease in horses caused by the protozoan parasites Sarcocystis neurona and Neospora hughesi.
- The diagnosis commonly relies on measuring antibody titers in serum and cerebrospinal fluid (CSF) using the indirect fluorescent antibody test (IFAT).
- Protein stability is critical for accurate IFAT titers, and long-term freezing storage can potentially alter proteins, affecting test reliability.
- Understanding the impact of prolonged sample storage at -80°C on IFAT results is important for both clinical diagnostics and research validity.
Study Objective
- To assess the effects of long-term storage (6 to 24 months) at -80°C on IFAT titers against S. neurona and N. hughesi in equine serum and CSF samples.
Methods
- Samples:
- 46 paired serum and CSF samples.
- 25 serum-only samples.
- Storage time intervals assessed:
- 6-12 months.
- 13-18 months.
- 19-24 months.
- Analysis:
- Antibody titers were compared across the storage periods against their fresh-testing baseline values.
- Statistical tests used include McNemar’s test and Wilcoxon rank tests to detect significant differences.
Key Results
- For Neospora hughesi:
- CSF samples stored for 19-24 months showed a significant difference in IFAT titers compared to fresh samples (p = 0.04), indicating an effect of long-term freezing on test results.
- No significant changes were observed in serum samples at any time point.
- For Sarcocystis neurona:
- No statistical differences were found in either serum or CSF samples at any storage interval (6-12, 13-18, or 19-24 months).
- Serum to CSF ratio (cutoff ≤ 64) used to support EPM diagnosis changed in about 41% (19 out of 46) of paired samples after long-term freezing, potentially impacting diagnostic interpretation.
Conclusions
- Long-term freezing at -80°C for more than 18 months can alter the IFAT results for N. hughesi in CSF, but not significantly for S. neurona.
- The alterations could affect the serum to CSF antibody ratio, which is often used diagnostically to confirm EPM.
- These changes may lead to misinterpretation of test results in both clinical and research settings if frozen samples are used without consideration of storage duration.
- It is recommended to be cautious interpreting IFAT titers from samples frozen beyond 18 months, especially for CSF testing related to N. hughesi.
Implications
- Clinical veterinarians should consider the storage time of frozen samples when relying on IFAT results for diagnosing EPM.
- Researchers using stored serum and CSF samples for serologic assays of EPM should account for potential degradation affecting antibody titers over extended freezing periods.
- Further studies may help optimize sample storage protocols or develop correction strategies for long-stored samples to improve diagnostic accuracy.
Cite This Article
APA
Valderrama-Martinez C, Packham A, Smith W, Mendoza-Flores JE, Zheng S, Chigerwe M, Plancarte M, Aleman M.
(2025).
Effect of Long-Term Freezing on Indirect Fluorescent Antibody Titers for the Diagnosis of Equine Protozoal Myeloencephalitis.
J Vet Intern Med, 39(5), e70225.
https://doi.org/10.1111/jvim.70225 Publication
Researcher Affiliations
- Departamento de Salud Animal, Facultad de Medicina Veterinaria y Zootecnia. Universidad Nacional de, Bogotá, Colombia.
- One Health Institute, School of Veterinary Medicine, University of California Davis, Davis, California, USA.
- One Health Institute, School of Veterinary Medicine, University of California Davis, Davis, California, USA.
- Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California Davis, Davis, California, USA.
- Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California Davis, Davis, California, USA.
- Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California Davis, Davis, California, USA.
- One Health Institute, School of Veterinary Medicine, University of California Davis, Davis, California, USA.
- Karen C. Drayer Wildlife Health Center, School of Veterinary Medicine, University of California Davis, Davis, California, USA.
- Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California Davis, Davis, California, USA.
MeSH Terms
- Animals
- Horses
- Horse Diseases / diagnosis
- Horse Diseases / parasitology
- Horse Diseases / blood
- Horse Diseases / cerebrospinal fluid
- Freezing
- Fluorescent Antibody Technique, Indirect / veterinary
- Encephalomyelitis / veterinary
- Encephalomyelitis / diagnosis
- Encephalomyelitis / parasitology
- Encephalomyelitis / cerebrospinal fluid
- Encephalomyelitis / blood
- Prospective Studies
- Antibodies, Protozoan / blood
- Antibodies, Protozoan / cerebrospinal fluid
- Sarcocystosis / veterinary
- Sarcocystosis / diagnosis
- Time Factors
- Neospora / immunology
Grant Funding
- PD1001 / The Seed for International Activities Program, sponsored by Global Affairs at UCD
- V435AM2 / Gifts from anonymous donors towards the Equine and Comparative Neurology Research Group
Conflict of Interest Statement
Authors declare no off‐label use of antimicrobials. Munashe Chigerwe serves as Associate Editor for the Journal of Veterinary Internal Medicine. He was not involved in the review of this manuscript. The other authors declare no conflicts of interest.
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