Optimization of a bovine cytokine multiplex assay using a new bovine and cross-reactive equine monoclonal antibodies.

Cytokines are important markers for immune activation, regulation, and homeostasis. The lack of monoclonal antibodies (mAbs) and sensitive assays to evaluate cytokine secretion has hindered research of bovine inflammation and immune regulation. We recently developed a fluorescent bead-based multiplex assay (multiplex assay) for bovine IL-10, TNF-α, and IFN-γ. Although the original assay covers a broad concentration range for the 3 targets, analytical sensitivity for IL-10 and IFN-γ could be improved to facilitate detection of these cytokines in their physiological low pg/mL range. To optimize the multiplex assay, we generated a new bovine IL-10 mAb and explored its use for the detection of intracellular and secreted bovine IL-10. The new bovine IL-10 mAb 130 recognized recombinant bovine IL-10 fusion protein and did not react with the fusion protein tag, or the TNF-α and IFN-γ standards in the multiplex assay. For improving IFN-γ detection, we explored cross-reactivity of anti-equine IFN-γ mAbs by intracellular staining of bovine stimulated peripheral blood mononuclear cells (PBMC). Equine IFN-γ mAb 3 showed excellent cross-reactivity with bovine IFN-γ by intracellular detection. Adding IL-10 mAb 130 and IFN-γ mAb 3 to the bovine multiplex assay substantially improved the analytical sensitivity with lower limits of detection in the low pg/mL range for all analytes. The detection ranges for the optimized multiplex assay were determined as 2 - 134,000 pg/mL for IL-10, 8 - 127,000 pg/mL for IFN-γ, and 12 - 193,000 pg/mL for TNF-α. The assay was next used to measure cytokine concentrations in cell culture supernatants from PBMC stimulated in plasma from whole blood stimulation to confirm native IL-10, TNF-α, and IFN-γ recognition and to explore the upper detection limits of the assay. In PBMC stimulation with a mix of phorbol myristate acetate (PMA) and ionomycin resulted in highest cytokine concentrations, while in plasma from whole blood stimulation, highest concentrations were observed in samples stimulated with a mix of lipopolysaccharide (LPS), phytohemagglutinin (PHA), and the TLR-2/6 agonist Pam2Csk4. PBMC and whole blood stimulation protocols showed that the optimized multiplex assay covers a wide linear detection range for measuring cytokine concentrations in bovine samples. For whole blood stimulation, a cocktail of pathogen associated molecular patterns elicited a stronger cytokine response than a mix of PMA and ionomycin, but response varied considerably between individual cattle. In conclusion, optimizing the bovine cytokine assay with new reagents improved the lower detection limits and widened the linear detection ranges while lowering the background of the multiplex assay.

Vaccination of horses with Lyme vaccines for dogs induces short-lasting antibody responses.

Borrelia burgdorferi can induce Lyme disease. Approved Lyme vaccines for horses are currently not available. In an effort to protect horses, veterinarians are using Lyme vaccines licensed for dogs. However, data to assess the response of horses to, or determine the efficacy of this off-label vaccine use are missing. Here, antibodies against outer surface protein A (OspA), OspC, and OspF were quantified in diagnostic serum submissions from horses with a history of vaccination with canine Lyme vaccines. The results suggested that many horses respond with low and often short-lasting antibody responses. Subsequently, four experimental vaccination trials were performed. First, we investigated antibody responses to three canine vaccines in B. burgdorferi-naïve horses. One killed bacterin vaccine induced antibodies against OspC. OspA antibodies were low for all three vaccines and lasted less than 16weeks. The second trial tested the impact of the vaccine dose using the OspA/OspC inducing bacterin vaccine in horses. A 2mL dose produced higher OspA and OspC antibody values than a 1mL dose. However, the antibody response again quickly declined, independent of dose. Third, the horses were vaccinated with 2 doses of a recombinant OspA vaccine. Previous vaccination and/or environmental exposure enhanced the magnitude and longevity of the OspA antibody response to about 20weeks. Last, the influence of intramuscular versus subcutaneous vaccine administration was investigated for the recombinant OspA vaccine. OspA antibody responses were not influenced by injection route. The current work highlights that commercial Lyme vaccines for dogs induce only transient antibody responses in horses which can also be of low magnitude. Protection from infection with B. burgdorferi should not be automatically assumed after vaccinating horses with Lyme vaccines for dogs.