Canine leptospirosis represents a diagnostic challenge to veterinarians, due to the variability in presenting clinical signs and interpretation of serology test results in dogs that have been vaccinated previously. None of the commercially available serological assays, including the microscopic agglutination test (MAT), have been verified to be capable of differentiating infected from vaccinated animals (DIVA). Recent work identified that half of primary practice attending dogs were up to date with their leptospirosis vaccination and would be expected to have circulating anti-leptospira antibodies (Taylor et al., 2022), indicating that this is a relevant issue for suspected leptospirosis cases in dogs in the UK. This study aimed to explore the utility of three leptospiral outer membrane proteins (OMPs: LipL32, LipL21 and LipL41) as potential DIVA targets in the luciferase immunoprecipitation system (LIPS) assay. N and C terminal nanoluciferase tagged recombinant proteins were generated for each OMP. Differences in reactivity between serum samples from MAT positive dogs (n = 29) and paired samples (n = 6 dogs) taken pre and 21 days post leptospirosis vaccination were assessed against these six constructs. Reactivity was greater towards the N terminal than the C terminal recombinant proteins for all three OMPs. None of the constructs appeared to demonstrate DIVA capability, although two (pNLF1-N-FLAG/LipL32 and pNLF1-N-FLAG/LipL21) were able to detect vaccine seroconversion. The findings of this work suggest that these particular OMP targets do not offer DIVA ability, however LipL32 and LipL21 may be suitable for use in immunoassays for vaccine trials or for detection of infections in humans, where there is no requirement for DIVA capability.

由于先前接种过疫苗的犬的临床症状和血清学检测结果的解释存在差异，犬钩端螺旋体病对兽医来说是一个诊断挑战。包括显微凝集试验 (MAT) 在内的市售血清学检测均未被证实能够区分感染动物和接种疫苗的动物 (DIVA)。最近的研究发现，一半的初级诊所主诊犬已接种了最新的钩端螺旋体病疫苗，并且预计将具有循环抗钩端螺旋体抗体（Taylor 等人，2022），这表明这对于疑似钩端螺旋体病病例来说是一个相关问题。在英国养狗。本研究旨在探讨三种钩端螺旋体外膜蛋白（OMP：LipL32、LipL21 和 LipL41）作为荧光素酶免疫沉淀系统 (LIPS) 测定中潜在 DIVA 靶标的效用。为每个 OMP 生成 N 和 C 末端纳米荧光素酶标记的重组蛋白。针对这六种构建体，评估了钩端螺旋体病疫苗接种前和接种后 21 天采集的 MAT 阳性狗 (n = 29) 和配对样本 (n = 6 只狗) 的血清样本之间反应性的差异。对于所有三种 OMP，N 端重组蛋白的反应性均高于 C 端重组蛋白。尽管两个构建体（pNLF1-N-FLAG/LipL32 和 pNLF1-N-FLAG/LipL21）能够检测疫苗血清转化，但似乎没有一个构建体表现出 DIVA 能力。这项工作的结果表明，这些特定的 OMP 靶点不提供 DIVA 能力，但是 LipL32 和 LipL21 可能适合用于疫苗试验或人类感染检测的免疫测定，而无需 DIVA 能力。